Constructs for expressing lysosomal polypeptides

ABSTRACT

Provided are isolated nucleic acids for expressing lysosomal polypeptides such as lysosomal acid α-glucosidase (GAA) and vectors comprising the same. In one embodiment, the invention provides an isolated nucleic acid encoding a chimeric polypeptide comprising a secretory signal sequence operably linked to a lysosomal polypeptide. In another representative embodiment, an isolated nucleic acid is provided comprising a coding region encoding a GAA and a GAA 3′ untranslated region (UTR), wherein the GAA 3′ UTR comprises a deletion therein. In another representative embodiment, the invention provides an isolated nucleic acid comprising a coding region encoding a GAA and a 3′ UTR, wherein the 3′ UTR is less than about 200 nucleotides in length and comprises a segment that is heterologous to the GAA coding region. Also provided are methods of making and using delivery vectors encoding lysosomal polypeptides, for example, to produce the lysosomal polypeptide or to treat subjects afflicted with a deficiency in the lysosomal polypeptide.

RELATED APPLICATION INFORMATION

This application claims the benefit of U.S. Provisional Application No.60/441,789, filed Jan. 22, 2003, which is incorporated by referenceherein in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel nucleic acid constructs encodinglysosomal polypeptides, in particular lysosomal acid α-glucosidase, aswell as methods of using the same to produce recombinant lysosomalpolypeptides and to treat lysosomal polypeptide deficiencies.

BACKGROUND OF THE INVENTION

Glycogen storage disease type II (GSD II) is a classical lysosomalstorage disorder, characterized by lysosomal accumulation of glycogenand tissue damage, primarily in muscle and heart (Hirschhorn, R. andReuser, A. J. (2001) In The Metabolic and Molecular Basis for InheritedDisease (Scriver, C. R., Beaudet, A. L., Sly, W. S. & Valle, D. Eds.),pp. 3389-3419. McGraw-Hill, New York). The underlying enzyme deficiencyis acid α-glucosidase (GAA). In severe, infantile GSD II progressivecardiomyopathy and myopathy lead to cardiorespiratory failure and deathby 1 year of age. In milder, juvenile and adult-onset GSD II,progressive weakness and respiratory failure are disabling and deathfrom respiratory failure occurs.

Animal models for human lysosomal acid α-glucosidase (hGAA) deficiencyaccurately mimic GSD II, and the efficacy of approaches to gene therapyfor GSD II can be evaluated in these systems. The GAA knockout (GAA-KO)mouse model accumulated glycogen in skeletal and cardiac muscle, anddeveloped weakness and reduced mobility (Raben, N., et al. (1998) J.Biol. Chem. 273:19086-19092, Bijvoet, A. G., et al. (1998) Hum. Mol.Genet. 7:53-62). The administration of recombinant GAA to a GAA-KO mousedemonstrated uptake of GAA by skeletal muscle, presumably throughreceptor-mediated uptake and delivery of GAA to the lysosomes (Bijvoet,A. G. et al. (1998) Hum. Mot. Genet. 7:1815-1824). The Japanese quailmodel is similar to juvenile-onset GSD II, and has been treatedsuccessfully with recombinant enzyme replacement (Kikuchi, T., et al.(1998) J. Ciin. Invest. 101:827-833). Enzyme therapy has demonstratedefficacy for severe, infantile GSD II; however the benefit of enzymetherapy is limited by the need for frequent infusions and thedevelopment of inhibitor antibodies against recombinant hGAA(Amalfitano, A., et al. (2001) Genet. In Med. 3:132-138). As analternative or adjunct to enzyme therapy, the feasibility of genetherapy approaches to treat GSD-II have been investigated (Amalfitano,A., et al. (1999) Proc. Natl. Acad. Sci. USA 96:8861-8866, Ding, E., etal. (2002) Mol. Ther. 5:436-446, Fraites, T. J., et al. (2002) Mol.Ther. 5:571-578, Tsujino, S., et al. (1998) Hum. Gene Ther.9:1609-1616).

Administration of an adenovirus (Ad) vector encoding hGAA that wastargeted to mouse liver in the GAA-KO mouse model reversed the glycogenaccumulation in skeletal and cardiac muscle within 12 days throughsecretion of hGAA from the liver and uptake in other tissues(Amalfitano, A., et al. (1999) Proc. Natl. Acad. Sci. USA 96:8861-8866).Antibodies against hGAA abbreviated the duration of hGAA secretion withan Ad vector in liver, although vector DNA and hGAA persisted in tissuesat reduced levels for many weeks (Ding, E., et al. (2002) Mol. Ther.5:436-446). Introduction of adeno-associate virus 2 (AAV2) vectorsencoding GAA normalized the GAA activity in the injected skeletal muscleand the injected cardiac muscle, and glycogen content was normalized inmuscle when an AAV1-pseudotyped vector was administered with improvedmuscle transduction (Fraites, T. J., et al. (2002) Mol. Ther.5:571-578). Muscle-targeted Ad vector gene therapy was attempted in theJapanese quail model, although only localized reversal of glycogenaccumulation at the site of vector injection was achieved (Tsujino. S.,et al. (1998) Hum. Gene Ther. 9:1609-1616).

Neonatal gene therapy may have greater efficacy than administrationlater in life, as evidenced by experiments in several rodent diseasemodels. An AAV vector administered intravenously on the second day oflife in β-glucoronidase deficient (Sly disease) mice producedtherapeutically relevant levels of β-glucoronidase and correctedlysosomal storage in multiple tissues, including liver and kidney (Daly,T. M., et al. (1999) Proc. Natl. Acad. Sci. USA 96:2296-2300).Similarly, intramuscular injection of the AAV vector produced sustained,therapeutic levels of expression of β-glucoronidase and eliminatedlysosomal storage in muscle and liver (Daly, T. M., et al. (1999) Hum.Gene Ther. 10:85-94). AAV vector DNA persisted in muscle and intransduced areas of the liver following neonatal intramuscular injectionin the Sly disease mouse (Daly, T. M., et al. (1999) Hum. Gene Ther.10:85-94). The number of AAV vector particles administered to neonatalSly mice was approximately 100-fold less than was needed to producetherapeutically relevant levels of proteins in adult mice (Kessler, P.D., et al. (1996) Proc. Natl. Acad. Sci. USA 93:14082-14087, Herzog, R.W., et al. (1997) Proc. Nat. Acad. Sci. USA 94:5804-5809, Nakai, H., etal. (1998) Blood 91:4600-4607, Snyder, R. O., et al. (1997) Nat. Genet.16:270-276, Koeberl, D. D., et al. (1999) Hum. Gene Ther. 10:2133-2140,Snyder, R. O., et al. (1999) Nat. Med. 5:64-70, Herzog, R. W. et al.(1999) Nat. Med. 5:56-63).

There is a need in the art for improved methods of producing lysosomalpolypeptides such as GAA in vitro and in vivo, for example, to treatlysosomal polypeptide deficiencies. Further, there is a need for methodsthat result in systemic delivery of GAA and other lysosomal polypeptidesto affected tissues and organs.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery of improvednucleic acid constructs for expressing lysosomal polypeptides such asGAA. One aspect of the invention provides an isolated nucleic acidencoding a chimeric polypeptide comprising a lysosomal polypeptideoperably linked to a secretory signal sequence such that targeting ofthe lysosomal polypeptide to the secretory pathway (i.e., instead of tothe lysosome) is enhanced. As another aspect, the invention encompassesisolated nucleic acids comprising a coding sequence for GAA and an“abbreviated” 3′ untranslated region (3′ UTR). The isolated nucleicacids of the invention can be used to produce recombinant lysosomalpolypeptides in vitro (e.g., in cultured cells) or in vivo (e.g., in ananimal or plant based protein production system or in methods oftherapeutic treatment). In addition, improved (i.e., higher) titers ofviral vectors encoding lysosomal polypeptides can be produced with theisolated nucleic acids of the invention.

Accordingly, as one aspect, the present invention provides an isolatednucleic acid encoding a chimeric polypeptide comprising a secretorysignal sequence operably linked to a lysosomal polypeptide (e.g., GAA).Also provided is the chimeric polypeptide comprising the secretorysignal sequence operably linked to the lysosomal polypeptide.

As another aspect, the present invention provides isolated nucleic acidscomprising a GAA coding sequence and an “abbreviated” 3′ UTR. In onerepresentative embodiment, the invention provides an isolated nucleicacid encoding a GAA, the isolated nucleic acid comprising: (a) a codingregion encoding a GAA, and (b) an abbreviated 3′ UTR, wherein the 3′untranslated region is a GAA 3′ UTR comprising a deletion therein (e.g.,a deletion of at least 25 consecutive nucleotides), so that uponintroduction into a cell, GAA polypeptide is produced at a higher levelfrom the isolated nucleic acid as compared with GAA polypeptideproduction from an isolated nucleic acid comprising a full-length GAA 3′UTR. In an exemplary embodiment, the 3′ UTR comprises a deletion in theregion shown as nucleotides 3301 through 3846 of SEQ ID NO:1 (FIG. 8).

In another representative embodiment, the invention provides an isolatednucleic acid encoding a GAA, the isolated nucleic acid comprising: (a) acoding region encoding a GAA, and (b) an abbreviated 3′ UTR, wherein the3′ UTR is less than about 200 nucleotides in length and comprises asegment that is heterologous to the GAA coding region, so that uponintroduction into a cell, GAA polypeptide is produced at a higher levelfrom the isolated nucleic acid as compared with GAA polypeptideproduction from an isolated nucleic acid comprising a full-length GAA 3′UTR.

In other particular embodiments, the lysosomal polypeptide is a humanpolypeptide and/or the isolated nucleic acid is operatively associatedwith a transcriptional control element that is operable in liver cells(optionally, with a liver-specific transcriptional control element).

As additional aspects, the present invention further provides vectors(including nonviral and viral vectors, the latter including adenovirus,AAV and hybrid adenovirus-AAV vectors), cells and pharmaceuticalformulations comprising the isolated nucleic acids of this invention.

As still further aspects, the present invention provides methods ofmaking delivery vectors (e.g., viral vectors) comprising the isolatednucleic acids of this invention.

Also provided are methods of using the isolated nucleic acids, vectors,cells, and pharmaceutical formulations of the invention to treatdeficiencies of lysosomal polypeptides (e.g., GAA) and to producerecombinant lysosomal polypeptides (e.g., in vitro in cultured cells orin vivo in an animal or plant-based recombinant protein expressionsystem or for therapeutic purposes).

In illustrative embodiments, the present invention is practiced toadminister an isolated nucleic acid encoding a lysosomal polypeptidesuch as GAA to a subject (for example, a subject diagnosed with orsuspected of having a deficiency of the lysosomal polypeptide). Inparticular representative embodiments, an isolated nucleic acid of theinvention encoding a lysosomal polypeptide can be administered to onedepot tissue or organ (e.g., liver, skeletal muscle, lung and the like)and the polypeptide expressed therein at levels sufficient to result insecretion into the systemic circulation, from which the secretedpolypeptide is taken up by distal target tissues (e.g., skeletal,cardiac and/or diaphragm muscle). Similarly, the isolated nucleic acidcan be delivered to brain cells (e.g., to treat MPS disorders such asSly disease), where the lysosomal polypeptide can be expressed, secretedand taken up by non-transformed or non-transduced brain cells (e.g.,neurons and glial cells).

In another particular embodiment, a recombinant adeno-associated virus(AAV) vector expressing an isolated nucleic acid encoding GAA of theinvention is administered to the liver of a subject having GAAdeficiency, which results in GAA polypeptide production at sufficientlyhigh levels such that GAA polypeptide is secreted from the liver andtaken up by skeletal muscle and/or other tissues, which advantageouslyleads to a reduction in glycogen content and/or an improvement in otherclinical indicia of GAA deficiency in affected tissues.

As yet a further aspect, the present invention provides the use of theisolated nucleic acids, vectors, cells and pharmaceutical formulationsof the invention in the manufacture of medicaments for the treatment oflysosomal polypeptide deficiencies (e.g., GAA deficiency).

These and other aspects of the invention are described in more detail inthe description of the invention set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Detection of hGAA following neonatal intramuscularadministration of an Ad-AAV vector in GAA-KO mice. (Panel A) Westernblot analysis of muscle after gastrocnemius of GAA-KO mice was injectedwith the Ad-AAV vector (4×10¹⁰ DRP) encoding hGAA at 3 days of age. Allsamples were obtained 24 weeks following vector administration.Recombinant human GAA (rhGAA) was the standard. Each lane for theindicated muscle groups represents one GAA-KO mouse. The ˜67 kD, ˜76 kD,and ˜110 kD hGAA species were detected in transduced muscle as expected(Amalfitano, A., et al. (1999) Proc. Natl. Acad. Sci. USA 96:8861-8866,Ding, E., et al. (2002) Mol. Ther. 5:436-446). (Panel B) Western blotanalysis of heart, liver and diaphragm following neonatal administrationof the Ad-AAV vector. Each lane represents one GAA-KO mouse analyzed atthe indicated time following vector administration. The samples forindividual mice were loaded in the same order for the Western blot ofeach tissue.

FIG. 2. GAA activity and glycogen content for skeletal muscle and othertissues in GAA-KO mice following neonatal Ad-AAV vector administration.(Panel A) The hGAA activity for gastrocnemius and quadriceps at 6, 12,and 24 weeks following vector administration, and for hamstrings at 24weeks, compared to the hGAA activity in gastrocnemius, Quadriceps, andhamstrings for untreated, GAA-KO mice. The average and standarddeviation are shown. The p value is indicated as follows: *<0.05,**<0.01, and ***<0.001. The number of mice (n) is shown for each timepoint. (Panel B) The hGAA activity for heart, liver, and diaphragm inmice following Ad-AAV vector administration, and for controls. Controlswere untreated, GAA-KO mice: n=4 for heart and liver, n=5 for diaphragm.The average and standard deviation are shown for controls.

FIG. 3. Antibodies in GAA-KO mouse plasma following neonatal Ad-AAVvector administration. (Panel A) The absorbance for anti-hGAA antibodiesin an ELISA of GAA-KO mouse plasma at 6 weeks following neonatal Ad-AAVvector administration (Neonatal Intramuscular Ad-AAV), and of GAA-KOmouse plasma at 6 weeks following intravenous Ad-AAV vectoradministration (4×10¹⁰ particles) in adult mice (Ad-AAV). Controlsconsisted of untreated, GAA-KO mice (Control). Each column representsthe mean and standard deviation for an individual mouse. Each dilutionof plasma (1:100, 1:200, and 1:400) was analyzed in triplicate. (PanelB) The titer for anti-hGAA antibodies by ELISA, representing the samemice in the same order as in FIG. 1 Panel B. Each sample was analyzed induplicate at each dilution. (Panel C) The absorbance for anti-Adantibodies in an ELISA of GAA-KO mouse plasma for the same samples asdescribed for the ELISA for anti-hGAA antibodies above, except that only2 untreated controls were analyzed. Each sample was analyzed induplicate at each dilution. The order of loading was the same as in FIG.1 Panel B.

FIG. 4. The glycogen content for skeletal muscle and heart. (Panel A)Gastrocnemius and quadriceps at 6, 12, and 24 weeks following vectoradministration, and for hamstrings at 24 weeks, compared to the hGAAactivity in gastrocnemius, quadriceps, and hamstrings for untreated,GAA-KO mice. The average and standard deviation are shown. The p valueis indicated as follows: *<0.05, **<0.01, and ***<0.001. The number ofmice (n) is shown for each time point. (Panel B) The glycogen contentfor heart and diaphragm, and for controls. Controls were untreated,GAA-KO mice (n=4). The average and standard deviation are shown forcontrols.

FIG. 5. Glycogen staining of skeletal muscle and heart. PAS stainingshowed glycogen accumulation in lysosomes, and pooling of glycogenoutside lysosomes, that was corrected following Ad-AAV administration atthe times indicated.

FIG. 6. hGAA synthesis (top panel) and glycogen content (lower panel) inmuscle of GAA-KO/SCID mice administered an AAV2/6 (AAV6) vectorexpressing GAA intramuscularly.

FIG. 7. Secretion of hGAA from liver into plasma in GAA-KO/SCID miceadministered an AAV2/2 (AAV2) or AAV2/6 (AAV6) vector. Western blotanalysis of plasma from GAA-KO/SCID mice following AAV vectoradministration, and from untreated GAA-KO/SCID mice (controls).

FIG. 8. A full-length hGAA cDNA sequence; Genebank Accession No.NM_000152 (SEQ ID NO:1). The encoded protein is shown in SEQ ID NO:2.The ORF is nt 442 . . . 3300. The GAA 3′ UTR sequence is from nt 3301 to3846, total 546 bp.

FIG. 9. A hGAA sequence with a deleted 3′ UTR (SEQ ID NO:3). The 411 bpfrom nt 3397 through to nt 3807 In the 3′ UTR of the sequence shown inFIG. 8 (SEQ ID NO:1) were deleted (bold, italic). Note that the polyAsignal (bold, 3825 . . . 3830) and polyA site (3846) are not deleted.The 5′ UTR sequence of nt 1 through nt 409 was also deleted.

FIG. 10. GAA activity in liver and other tissues following portal veininjection of an AAV2/2 vector. GAA-KO/SCID mice received the vectorpackaged as AAV2 (AAV2/2) at 3 months of age (n=3), and were analyzed 12weeks after injection. Controls were 3 month-old, untreated GAA-KO/SCIDmice (n=3).

FIG. 11. Human GAA in liver and other tissues following portal veininjection of AAV vectors in GAA-KO/SCID mice. Western blot analysis ofplasma from GAA-KO/SCID mice at the indicated times following AAV vectoradministration, and from untreated GAA-KO/SCID mice (controls).Recombinant hGAA (rhGAA) is shown as a standard.

FIG. 12. GAA activity in gastrocnemius muscles (injected and uninjected)and other tissues following intramuscular injection of an AAV2/6 vector.GAA-KO/SCID mice received the vector packaged as AAV 6 (AAV2/6) at 6weeks of age (n=8), and were analyzed at 6, 12 and 24 weeks afterinjection. Controls were 3 month-old, untreated GAA-KO/SCID mice (n=3).

FIG. 13. Glycogen staining of skeletal muscle in GAA-KO/SCID mice. PASstaining showed glycogen accumulation in lysosomes, and pooling ofglycogen outside lysosomes that was corrected following AAV2/6administration.

FIG. 14. Localization (cellular vs. secreted) of GAA with various leadersequences expressed in transfected 293 cells.

FIG. 15. Western biot analysis of GAA with various leader sequences. GAAexpressed in transfected 293 cells with the constructs containing hGAAlinked to the indicated signal peptides. For transfected 293 cells, inlanes 3-14, the first (odd-numbered) of 2 lanes for each constructrepresents the cell lysate and the second lane (even-numbered) is themedium. The control represents untransfected 293 cells, showingendogenous, processed hGAA. Recombinant human GAA (rhGAA) is shown as astandard.

FIG. 16. Western blot analysis of plasma from GAA-KO/SCID mice at 2weeks following vector administration, and from untreated GAA-KO/SCIDmice (controls). Three-month-old GAA-KO/SCID mice received an AAV vectorencoding the chimeric α-1-antitrypsin signal peptide linked to the hGAAcDNA (Alpha-1-antitrypsin, lanes 2-7), or an AAV vector encoding hGAAwith its endogenous signal peptide (hGAA, lanes 8-13). Each lanerepresents an individual mouse. Lanes 5-7 and 11-13 were female mice.Recombinant human GAA (rhGAA) is shown as a standard.

FIG. 17. GAA activity in liver and other tissues at 2 weeks followingintravenous injection of an AAV2/8 vector encoding hGAA linked to thealpha-1-antitrypsin leader sequence. Male 3 month-old GAA-KO/SCID mice(n=3) received an AAV vector encoding the chimeric alpha-1-antitrypsinsignal peptide linked to the hGAA cDNA (minus the 27 amino acid GAAsignal peptide). Controls were untreated GAA-KO/SCID mice (n=3).

FIG. 18. Human GAA in liver and other tissues following intravenousinjection of an AAV vector containing a liver-specific promoter to driveGAA expression in immunocompetent GAA-KO mice. Western blot analysis ofplasma from GAA-KO mice at 3 weeks following AAV vector administration,and from untreated GAA-KO mice (controls). Recombinant human rhGAA(rhGAA) is shown as a standard.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is based, in part, on the discovery of improvednucleic acid constructs for expressing lysosomal polypeptides such asGAA. One aspect of the invention provides an isolated nucleic acidencoding a chimeric polypeptide comprising a lysosomal polypeptideoperably linked to a secretory signal sequence such that targeting ofthe lysosomal polypeptide to the secretory pathway (i.e., instead of tothe lysosome) is enhanced. As another aspect, the invention encompassesisolated nucleic acids comprising a coding sequence for GAA and an“abbreviated” 3′ UTR.

The isolated nucleic acids of the invention are advantageous fordelivery of lysosomal polypeptides (e.g., GAA) to target cells or forrecombinant protein production in cultured cells or tissues or wholeanimal systems (e.g., for enzyme replacement therapy). In particularembodiments, the present invention can be practiced to deliver a codingsequence for a lysosomal polypeptide to a “depot” tissue or organ (e.g.,liver, skeletal muscle, lung), where the polypeptide is expressed in thedepot tissue or organ, secreted into the systemic circulation, and takenup by target tissues (e.g., skeletal muscle, cardiac muscle and/ordiaphragm muscle in GAA deficient individuals). In representativeembodiments, uptake of GAA polypeptide secreted from the liver byskeletal muscle and/or other tissues affected by GAA deficiency resultsin a reduction in glycogen stores or improvement in other clinicalindicia of GAA deficiency. In other embodiments, the isolated nucleicacid is delivered to cells (e.g., neurons and/or glial cells) in thebrain, the lysosomal polypeptide is produced and secreted by thetransformed or transduced cells and taken up by other brain cells.

The present invention will now be described with reference to theaccompanying drawings, in which preferred embodiments of the inventionare shown. The terminology used in the description of the inventionherein is for the purpose of describing particular embodiments only andis not intended to be limiting of the invention. This invention may beembodied in different forms and should not be construed as limited tothe embodiments set forth herein. Rather, these embodiments are providedso that this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art. Forexample, features illustrated with respect to one embodiment may beincorporated into other embodiments, and features illustrated withrespect to a particular embodiment may be deleted from that embodiment.In addition, numerous variations and additions to the embodimentssuggested herein will be apparent to those skilled in the art in lightof the instant disclosure, which do not depart from the instantinvention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Except as otherwise indicated, standard methods may be used for theproduction of viral and non-viral vectors, manipulation of nucleic acidsequences, production of transformed cells, recombinant proteinproduction, and the like according to the present invention. Suchtechniques are known to those skilled in the art. See, e.g., SAMBROOK etal., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor,N.Y., 1989); F. M. AUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY(Green Publishing Associates, Inc. and John Wiley & Sons, Inc., NewYork).

Definitions

Unless indicated otherwise, explicitly or by context, the followingterms are used herein as set forth below:

As used in the description of the invention and the appended claims, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

As used herein, a “vector” or “delivery vector” may be a viral ornon-viral vector that is used to deliver a nucleic acid to a cell,tissue or subject.

A “recombinant” vector or delivery vector refers to a viral or non-viralvector that comprises one or more heterologous nucleotide sequences(i.e., transgenes). e.g., two, three, four, five or more heterologousnucleotide sequences. The recombinant vectors of the invention comprisenucleotide sequences that encode GAA, but may also comprise one or moreadditional heterologous sequences.

As used herein, the term “viral vector” or “viral delivery vector” mayrefer to a virus particle that functions as a nucleic acid deliveryvehicle, and which comprises the recombinant vector genome packagedwithin a virion. Alternatively, these terms may be used to refer to thevector genome when used as a nucleic acid delivery vehicle in theabsence of the virion.

A viral “vector genome” refers to the viral genomic DNA or RNA, ineither its naturally occurring or modified form. A “recombinant vectorgenome” is a viral genome (e.g., vDNA) that comprises one or moreheterologous nucleotide sequence(s).

A “heterologous nucleotide sequence” will typically be a sequence thatis not naturally-occurring in the vector. Alternatively, a heterologousnucleotide sequence may refer to a sequence that is placed into anon-naturally occurring environment (e.g., by association with apromoter with which it is not naturally associated).

By “infectious,” as used herein, it is meant that a virus can enter acell by natural transduction mechanisms and express viral genes(including heterologous nucleotide sequence(s)). Alternatively, an“infectious” virus is one that can enter the cell by other mechanismsand express the genes encoded by the viral genome. As one illustrativeexample, the vector can enter a target cell by expressing a ligand orbinding protein for a cell-surface receptor in the virion or by using anantibody(ies) directed against molecules on the cell-surface followed byinternalization of the complex.

As used herein, “transduction” of a cell by AAV means that the AAVenters the cell to establish a latent infection. See, e.g., BERNARD N.FIELDS et al., VIROLOGY, volume 2, chapter 69 (3d ed., Lippincott-RavenPublishers).

The term “replication” as used herein in reference to viral vectorsrefers specifically to replication (i.e., making new copies of) of thevector genome (i.e., virion DNA or RNA).

The term “propagation” as used herein in reference to viral vectorsrefers to a productive viral infection wherein the viral genome isreplicated and packaged to produce new virions, which typically can“spread” by infection of cells beyond the initially infected cell. A“propagation-defective” virus is impaired in its ability to produce aproductive viral infection and spread beyond the initially infectedcell.

As used herein, the term “polypeptide” encompasses both peptides andproteins, unless indicated otherwise.

A “chimeric polypeptide” is a polypeptide produced when two heterologousgenes or fragments thereof coding for two (or more) differentpolypeptides or fragments thereof not found fused together in nature arefused together in the correct translational reading frame. Illustrativechimeric polypeptides include fusions of GAA or other lysosomalpolypeptides to all or a portion of glutathione-S-transferase,maltose-binding protein, or a reporter protein (e.g., Green FluorescentProtein, β-glucuronidase, and β-galactosidase, luciferase). Inparticular embodiments of the invention, the chimeric polypeptidecomprises a secretory signal sequence operably linked to a lysosomalpolypeptide (e.g., GAA).

As used herein, a “functional” polypeptide is one that retains at leastone biological activity normally associated with that polypeptide.Preferably, a “functional” polypeptide retains all of the activitiespossessed by the unmodified polypeptide. By “retains” biologicalactivity, it is meant that the polypeptide retains at least about 50%,60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biologicalactivity of the native polypeptide (and may even have a higher level ofactivity than the native polypeptide). A “non-functional” polypeptide isone that exhibits essentially no detectable biological activity normallyassociated with the polypeptide (e.g., at most, only an insignificantamount, e.g., less than about 10% or even 5%).

As used herein, an “isolated” nucleic acid (e.g., an “isolated DNA” oran “isolated vector genome”) means a nucleic acid separated orsubstantially free from at least some of the other components of thenaturally occurring organism or virus, for example, the cell or viralstructural components or other polypeptides or nucleic acids commonlyfound associated with the nucleic acid.

Likewise, an “isolated” polypeptide means a polypeptide that isseparated or substantially free from at least some of the othercomponents of the naturally occurring organism or virus, for example,the cell or viral structural components or other polypeptides or nucleicacids commonly found associated with the polypeptide.

A “therapeutically effective” amount as used herein is an amount that issufficient to provide some improvement or benefit to the subject.Alternatively stated, a “therapeutically-effective” amount is an amountthat will provide some alleviation, mitigation or decrease in at leastone clinical symptom in the subject. To illustrate, in the case of GAAdeficiency, an amount that provides some alleviation, mitigation ordecrease in at least one clinical symptom of GAA deficiency (e.g.,reduced glycogen stores in skeletal, diaphragm and/or cardiac muscle,improved muscle strength and function, improved pulmonary function,improved motor development or attainment of motor developmentalmilestones, reduction in need for or prevention of need for ventilatorsupport, prevention of cardiac or cardiorespiratory failure, reducedpremature mortality, and the like). Those skilled in the art willappreciate that the therapeutic effects need not be complete orcurative, as long as some benefit is provided to the subject.

By the terms “treating” or “treatment of,” it is intended that theseverity of the patient's condition is reduced or at least partiallyimproved and that some alleviation, mitigation, delay or decrease in atleast one clinical symptom is achieved.

A “reduction in glycogen stores” in a tissue is intended to indicateabout a 25%, 35%, 40%, 50%, 60%, 75%, 85%, 90% 95% or more reduction intotal glycogen in a particular tissue, unless otherwise indicated (e.g.,a reduction in lysosomal glycogen stores or in pooled tissues).

By the term “express” or “expression” of a nucleic acid coding sequence,in particular a GAA coding sequence, it is meant that the sequence istranscribed, and optionally, translated. Generally, however, accordingto the present invention, the term “express” or “expression” is intendedto refer to transcription and translation of the coding sequenceresulting in production of the encoded polypeptide.

By “enhanced” or “enhancement” (or grammatical variations thereof) withrespect to nucleic acid expression or polypeptide production, it ismeant an increase and/or prolongation of steady-state levels of theindicated nucleic acid or polypeptide, e.g., by at least about 20%, 25%,40%, 50%, 60%, 75%, 2-fold, 2.5-fold, 3-fold, 5-fold, 10-fold, 15-fold,20-fold, 30-fold, 50-fold, 100-fold or more.

Unless indicated otherwise, the terms “enhanced” or “enhancement” (orgrammatical variations thereof) with respect to polypeptide secretionindicates an increase in the relative proportion of polypeptide that issecreted from the cell, e.g., by at least about 20%, 25%, 40%, 50%, 60%,75%, 2-fold, 2.5-fold, 3-fold, 5-fold, 10-fold, 15-fold, 20-fold,30-fold, 50-fold, 100-fold or more.

II. Improved Constructs for Producing Lysosomal Polypeptides

As described in more detail below, the present invention providesimproved constructs for producing lysosomal polypeptides, e.g.,polypeptides that are targeted to the lysosomes. As known in the art,many lysosomal proteins are characterized by the presence ofmannose-6-phosphate residues, and in embodiments of the invention thelysosomal polypeptide comprises mannose-6-phosphate glycosylation. Inother representative embodiments, the lysosomal polypeptide is one thatis associated with a lysosomal storage disease (e.g., because of adeficiency or defect in the lysosomal polypeptide). By “associated witha lysosomal storage disease”, it is meant that the lysosomal polypeptideis one that is deficient or defective in a lysosomal storage disorder,or is otherwise a causative agent in a lysosomal storage disorder.

As known in the art, there are a multitude of lysosomal storagediseases. Exemplary lysosomal storage disease include, but are notlimited to, glycogen storage disease type II (GSD II or Pompe Disease).GM1 gangliosidosis, Tay-Sachs disease, GM2 gangliosidosis (AB variant),Sandhoff disease, Fabry disease, Gaucher disease, metachromaticleukodystrophy, Krabbe disease, Niemann-Pick disease (Types A-D). Farberdisease, Wolman disease, Hurler Syndrome (MPS III), Scheie Syndrome (MPSIS), Hurer-Scheie Syndrome (MPS IH/S), Hunter Syndrome (MPS II),Sanfilippo A Syndrome (MPS IIIA), Sanfilippo B Syndrome (MPS IIIB),Sanfilippo C Syndrome (MPS IIIC), Sanfilippo D Syndrome (MPS IIID),Morquio A disease (MPS IVA), Morquio B disease (MPS IV B),Maroteaux-Lamy disease (MPS VI), Sly Syndrome (MPS VII), α-mannosidosis,β-mannosidosis, fucosidosis, aspartylglucosaminuria, sialidosis(mucolipidosis I), mucolipidosis II (I-Cell disease), mucolipidosis III(pseudo-Hurler polydystrophy), mucolipidosis IV, galactosialidosis(Goldberg Syndrome), Schindler disease, cystinosis, Salla disease,infantile sialic acid storage disease, Batten disease (juvenile neuronalceroid lipofuscinosis), infantile neuronal ceroid lipofuscinosis, andprosaposin.

Lysosomal polypeptides that are associated with lysosomal storagediseases according to the present invention include, but are not limitedto, lysosomal acid α-glucosidase (GAA; also known as acid maltase),α-galactosidase A, β-galactosidase, β-hexosaminidase A, β-hexosaminidaseB, GM₂ activator protein, glucocerebrosidase, arylsulfatase A,galactosylceramidase, acid sphingomyelinase, acid ceramidase, acidlipase, α-L-iduronidase, iduronate sulfatase, heparan N-sulfatase,α-N-acetylglucosaminidase, glucosaminide acetyltransferase,N-acetylglucosamine-6-sulfatase, arylsulfatase B, β-glucuronidase,α-mannosidase, β-mannosidase, α-L-fucosidase,N-aspartyl-β-glucosaminidase, N-acetylgalactosamine 4-sulfatase,α-neuraminidase, lysosomal protective protein,α-N-acetyl-galactosaminidase, N-acetylglucosamine-1-phosphotransferase,cystine transport protein, sialic acid transport protein, the CLN3 geneproduct, palmitoyl-protein thioesterase, saposin A, saposin B, saposinC. and saposin D.

Lysosomal acid α-glucosidase or “GAA” (E.C. 3.2.1.20) (1,4-α-D-glucanglucohydrolase), is an exo-1,4-α-D-glucosidase that hydrolyses bothα-1,4 and α-1,6 linkages of oiigosaccharides to liberate glucose. Adeficiency in GAA results in glycogen storage disease type II (GSDII),also referred to as Pompe disease (although this term formally refers tothe infantile onset form of the disease). It catalyzes the completedegradation of glycogen with slowing at branching points. The 28 kbhuman acid α-glucosidase gene on chromosome 17 encodes a 3.6 kb mRNAwhich produces a 951 amino acid polypeptide (Hoefsloot et al., (1988)EMBO J. 7:1697; Martiniuk et al., (1990) DNA and Cell Biology 9:85). Theenzyme receives co-translational N-linked glycosylation in theendoplasmic reticulum. It is synthesized as a 110-kDa precursor form,which matures by extensive glycosylation modification, phosphorylationand by proteolytic processing through an approximately 90-kDa endosomalintermediate into the final lysosomal 76 and 67 kDa forms (Hoefsloot,(1988) EMBO J. 7:1697; Hoefsloot et al., (1990) Biochem. J. 272:485;Wisselaar et al., (1993) J. Biol. Chem. 268:2223; Hermans et al., (1993)Biochem. J. 289:681).

In patients with GSD II, a deficiency of acid α-glucosidase causesmassive accumulation of glycogen in lysosomes, disrupting cellularfunction (Hirschhorn, R. and Reuser, A. J. (2001), in The Metabolic andMolecular Basis for Inherited Disease, (eds, Scriver, C. R. et al.)pages 3389-3419 (McGraw-Hill, New York). In the most common infantileform, patients exhibit progressive muscle degeneration andcardiomyopathy and die before two years of age. Severe debilitation ispresent in the juvenile and adult onset forms.

The term “GAA” or “GAA polypeptide,” as used herein, encompasses mature(˜76 or ˜67 kDa) and precursor (e.g., ˜110 kDa) GAA as well as modified(e.g., truncated or mutated by insertion(s), deletion(s) and/orsubstitution(s)) GAA proteins or fragments thereof that retainbiological function (i.e., have at least one biological activity of thenative GAA protein, e.g. can hydrolyze glycogen, as defined above) andGAA variants (e.g., GAA II as described by Kunita et al., (1997)Biochemica et Biophysica Acta 1362.269: GAA polymorphisms and SNPs aredescribed by Hirschhorn. R. and Reuser. A. J. (2001) in The Metabolicand Molecular Basis for Inherited Disease (Scriver, C. R., Beaudet, A.L., Sly, W. S. & Valle, D. Eds.), pp. 3389-3419. McGraw-Hill, New York,see pages 3403-3405; each incorporated herein by reference in itsentirety). Any GAA coding sequence known in the art may be used, forexample, see the coding sequences of FIGS. 8 and 9; GenBank Accessionnumber NM_00152 and Hoefsloot et al., (1988) EMBO J. 7:1697 and Van Hoveet al., (1996) Proc. Natl. Acad. Sci. USA 93:65 (human), GenBankAccession number NM_008064 (mouse), and Kunita et al., (1997) Biochemicaet Biophysica Acta 1362:269 (quail); the disclosures of which areincorporated herein by reference for their teachings of GAA coding andnoncoding sequences.

Likewise, the term “lysosomal polypeptide,” as used herein, encompassesmature and precursor lysosomal polypeptides as well as modified (e.g.,truncated or mutated by insertion(s), deletion(s) and/orsubstitution(s)) lysosomal polypeptides or fragments thereof that retainbiological function (i.e., have at least one biological activity of thenative lysosomal polypeptide) and lysosomal polypeptide variants.

The coding sequence of the lysosomal polypeptide can be derived from anysource, including avian and mammalian species. The term “avian” as usedherein includes, but is not limited to, chickens, ducks, geese, quail,turkeys and pheasants. The term “mammal” as used herein includes, but isnot limited to, humans, simians and other non-human primates, bovines,ovines, caprines, equines, felines, canines, lagomorphs, etc. Inembodiments of the invention, the nucleic acids of the invention encodea human, mouse or quail lysosomal polypeptide.

A. Constructs for Targeting Lysosomal Polypeptides to the SecretoryPathway.

Lysosomal proteins generally have amino-terminal signal peptides thatco-translationally transfer the nascent proteins to the lumen of theendoplasmic reticulum. It is believed that lysosomal polypeptidesdiverge from the secretory pathway and are directed to the lysosome byat least three distinct pathways (see. e.g., Wisselaar et al., (1993) J.Biol. Chem. 268:2223-31). The best studied of these involves thepost-translational addition of mannose-6-phosphate residues that arerecognized by the phosphomannosyl receptor, which directs the transportof the polypeptide to the lysosome.

The first 27 amino acids of the human GAA polypeptide are typical ofsignal peptides of lysosomal and secretory proteins. GAA may be targetedto lysosomes via the phosphomannosyl receptor and/or by sequencesassociated with the delayed cleavage of the signal peptide (Hirschhom,R. and Reuser, A. J. (2001), in The Metabolic and Molecular Basis forInherited Disease, (eds, Scriver, C. R. et al.) pages 3389-3419(McGraw-Hill, New York). A membrane-bound precursor form of the enzyme(i.e., anchored by the uncleaved signal peptide) has been identified inthe lumen of the endoplasmic reticulum (see, e.g., Wisselaar et al.,(1993) J. Biol. Chem. 268:2223-31).

The present invention provides isolated nucleic acids encoding lysosomalpolypeptides (e.g., GAA) that are fused to a signal peptide thatenhances targeting of the polypeptide to the secretory pathway.Secretion of lysosomal polypeptides from the cell provides a number ofadvantages. For example, in recombinant protein production systems (bothcultured cells/tissues or whole animals systems), it is generallypreferable to purify a secreted polypeptide from the extracellularmedium or fluids rather than harvesting the cells and isolatingintracellular protein. With respect to therapeutic methods, it has beenshown that administration of an Ad vector encoding hGAA that wastargeted to liver in a GAA knock-out mouse model reversed glycogenaccumulation in skeletal and cardiac muscle by secretion of hGAA fromthe liver and uptake by affected tissues (Amalfitano et al., Proc. Nat.Acad. Sci. USA 96:8861-8866). Presumably, secretion of significantamounts of GAA (i.e., rather than lysosomal targeting) was a result ofoverexpression of the GAA transgene delivered by the Ad vector andsaturation of the “scavenger” system that normally redirectsextracellular lysosomal proteins to the lysosome.

The present invention advantageously provides improved constructs thatenhance secretion of lysosomal polypeptides from the transduced ortransfected cell. These constructs facilitate the use of alternativedelivery systems (e.g., AAV vectors for liver delivery), enhance thesecretion of lysosomal polypeptides (e.g., for in vivo gene delivery orin vitro enzyme production), and can reduce or avoid cytotoxicity ororgan toxicity (e.g., hepatotoxicity) that may result fromover-accumulation of recombinant protein in the depot organ.

Accordingly, the invention encompasses isolated nucleic acids encoding achimeric polypeptide comprising a secretory signal sequence operablylinked to a lysosomal polypeptide (e.g., GAA) as well as the chimericpolypeptides. The secretory signal sequence is foreign to (e.g.,exogenous to) the lysosomal polypeptide. While those skilled in the artwill appreciate that secretory signal sequences are typically at theamino-terminus of the nascent polypeptide, the secretory signal sequenceaccording to the present invention can be located at any position withinthe chimeric polypeptide (e.g., N-terminal, within the maturepolypeptide, or C-terminal) as long as it functions as a secretorysignal sequence (e.g., enhances secretion of the lysosomal polypeptide)and does not render the lysosomal polypeptide non-functional.

As used herein, the term “secretory signal sequence” or variationsthereof are intended to refer to amino acid sequences that function toenhance (as defined above) secretion of an operably linked lysosomalpolypeptide from the cell as compared with the level of secretion seenwith the native lysosomal polypeptide. As defined above, by “enhanced”secretion, it is meant that the relative proportion of lysosomalpolypeptide synthesized by the cell that is secreted from the cell isincreased; it is not necessary that the absolute amount of secretedprotein is also increased. In particular embodiments of the invention,essentially all (i.e., at least 95%, 97%, 98%, 99% or more) of thepolypeptide is secreted. It is not necessary, however, that essentiallyall or even most of the lysosomal polypeptide is secreted, as long asthe level of secretion is enhanced as compared with the native lysosomalpolypeptide.

In particular embodiments, at least about 50%, 60%, 75% 85%, 90%, 95%,98% or more of the lysosomal polypeptide is secreted from the cell.

The relative proportion of newly-synthesized lysosomal polypeptide thatis secreted from the cell can be routinely determined by methods knownin the art and as described in the Examples. Secreted proteins can bedetected by directly measuring the protein itself (e.g., by Westernblot) or by protein activity assays (e.g., enzyme assays) in cellculture medium, serum, milk, etc.

Generally, secretory signal sequences are cleaved within the endoplasmicreticulum and, in particular embodiments of the invention, the secretorysignal sequence is cleaved prior to secretion. It is not necessary,however, that the secretory signal sequence is cleaved as long assecretion of the lysosomal polypeptide from the cell is enhanced and thelysosomal polypeptide is functional. Thus, in embodiments of theinvention, the secretory signal sequence is partially or entirelyretained.

Thus, in particular embodiments of the invention, an isolated nucleicacid encoding a chimeric polypeptide comprising a lysosomal polypeptideoperably linked to a secretory signal sequence is delivered to a cell,and the chimeric polypeptide is produced and the lysosomal polypeptidesecreted from the cell. The lysosomal polypeptide can be secreted aftercleavage of all or part of the secretory signal sequence. Alternatively,the lysosomal polypeptide can retain the secretory signal sequence(i.e., the secretory signal is not cleaved). Thus, in this context, the“lysosomal polypeptide” can be a chimeric polypeptide.

Those skilled in the art will further understand that the chimericpolypeptide can contain additional amino acids, e.g., as a result ofmanipulations of the nucleic acid construct such as the addition of arestriction site, as long as these additional amino acids do not renderthe secretory signal sequence or the lysosomal polypeptidenon-functional. The additional amino acids can be cleaved or can beretained by the mature polypeptide as long as retention does not resultin a nonfunctional polypeptide.

In representative embodiments, the secretory signal peptide replacesmost, essentially all or all of the leader sequence found in the nativelysosomal polypeptide. In particular embodiments, most or all of thenative leader sequence is retained, as long as secretion of thelysosomal polypeptide is enhanced and the mature lysosomal polypeptideis functional.

The secretory signal sequence can be derived in whole or in part fromthe secretory signal of a secreted polypeptide (i.e., from theprecursor) and/or can be in whole or in part synthetic. The secretorysignal sequence can be from any species of origin, including animals(e.g., avians and mammals such as humans, simians and other non-humanprimates, bovines, ovines, caprines, equines, porcines, canines,felines, rats, mice, lagomorphs), plants, yeast, bacteria, protozoa orfungi. The length of the secretory signal sequence is not critical;generally, known secretory signal sequences are from about 10-15 to50-60 amino acids in length. Further, known secretory signals fromsecreted polypeptides can be altered or modified (e.g., by substitution,deletion, truncation or insertion of amino acids) as long as theresulting secretory signal sequence functions to enhance secretion of anoperably linked lysosomal polypeptide.

The secretory signal sequences of the invention can comprise, consistessentially of or consist of a naturally occurring secretory signalsequence or a modification thereof (as described above). Numeroussecreted proteins and sequences that direct secretion from the cell areknown in the art. Exemplary secreted proteins (and their secretorysignals) include but are not limited to: erythropoietin, coagulationFactor IX, cystatin, lactotransferrin, plasma protease C1 inhibitor,apolipoproteins (e.g., APO A, C, E), MCP-1, α-2-HS-glycoprotein,α-1-microgolubilin, complement (e.g., C1Q, C3), vitronectin,lymphotoxin-α, azurocidin, VIP, metalloproteinase inhibitor 2,glypican-1: pancreatic hormone, clusterin, hepatocyte growth factor,insulin. α-1-antichymotrypsin, growth hormone, type IV collagenase,guanylin, properdin, proenkephalin A, inhibin β (e.g., A chain),prealbumin. angiogenin, lutropin (e.g., β chain), insulin-like growthfactor binding protein 1 and 2, proactivator polypeptide, fibrinogen(e.g., 1 chain), gastric triacylglycerol lipase midkine neutropnildefensins 1, 2, and 3, α-1-antitrypsin, matrix gia-protein. α-tryptase,bile-salt-activated lipase, chymotrypsinogen B, elastin, IG lambda chainV region, platelet factor 4 variant, chromogranin A, WNT-1proto-oncogene protein, oncostatin M, β-neoendorphin-dynorphin, vonWillebrand factor, plasma serine protease inhibitor, serum amyloid Aprotein, nidogen, fibronectin, rennin, osteonectin, histatin 3,phospholipase A2, cartilage matrix protein, GM-CSF, matrilysin, MIP-2-β,neuroendocrine protein 7B2, placental protein 11, gelsolin, IGF 1 and 2,M-CSF, transcobalamin 1, lactase-phlonzin hydrolase, elastase 2B,pepsinogen A, MIP 1-β, prolactin, trypsinogen II, gastrin-releasingpeptide II, atrial natriuretic factor, secreted alkaline phosphatase,pancreatic α-amylase, secretogranin I, β-casein, serotransferrin, tissuefactor pathway inhibitor, follitropin β-chain, coagulation factor XII,growth hormone-releasing factor, prostate seminal plasma protein,interleukins (e.g., 2, 3, 4, 5, 9, 11), inhibin (e.g., alpha chain),angiotensinogen, thyroglobulin, IG heavy or light chains, plasminogenactivator inhibitor-1, lysozyme C, plasminogen activator,antileukoproteinase 1, statherin, fibulin-1, isoform B, uromodulin,thyroxine-binding globulin, axonin-1, endometrial α-2 globulin,interferon (e.g., alpha, beta, gamma), β-2-microglobulin,procholecystokinin, progastricsin, prostatic acid phosphatase, bonesialoprotein II, colipase, Alzheimer's amyloid A4 protein, PDGF (e.g., Aor B chain), coagulation factor V, triacyiglycerol lipase,haptoglobuin-2, corticosteroid-binding globulin, triacylglycerol lipase,prorelaxin H2, follistatin 1 and 2, platelet glycoprotein IX, GCSF,VEGF, heparin cofactor II, antithrombin-II, leukemia inhibitory factor,interstitial collagenase, pieiotrophin, small inducible cytokine A1,melanin-concentrating hormone, angiotensin-converting enzyme, pancreatictrypsin inhibitor, coagulation factor VIII, α-fetoprotein,α-lactalbumin, senogelin II, kappa casein, glucagon, thyrotropin betachain, transcobalamin II, thrombospondin 1, parathyroid hormone,vasopressin copeptin, tissue factor, motilin, MPIF-1, kininogen,neuroendocrine convertase 2, stem cell factor procollagen α1 chain,plasma kallikrein, keratinocyte growth factor, as well as any othersecreted hormone, growth factor, cytokine, enzyme, coagulation factor,milk protein, immunoglobulin chain, and the like.

In other particular embodiments, the secretory signal sequence isderived in part or in whole from a secreted polypeptide that is producedby liver cells.

The secretory signal sequence of the invention can further be in wholeor in part synthetic or artificial. Synthetic or artificial secretorysignal peptides are known in the art, see e.g., Barash et al., “Humansecretory signal peptide description by hidden Markov model andgeneration of a strong artificial signal peptide for secreted proteinexpression,” Biochem. Biophys. Res. Comm. 294:835-42 (2002); thedisclosure of which is incorporated herein in its entirety. Inparticular embodiments, the secretory signal sequence comprises,consists essentially of, or consists of the artificial secretory signal:MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO: 5) or variations thereof having 1, 2,3, 4, or 5 amino acid substitutions (optionally, conservative amino acidsubstitutions, conservative amino acid substitutions are known in theart).

The isolated nucleic acid encoding the chimeric polypeptide can furthercomprise an “abbreviated” 3′ UTR as described in more detail below.

B. “Abbreviated” GAA Constructs.

The present invention is based, in part, on the discovery thatconstructs expressing lysosomal acid α-glucosidase (GAA) that aredeleted or altered (e.g., substituted) in the 3′ untranslated region(UTR), can have advantageous properties as compared with non-deleted ornon-altered constructs. For example, the efficiency of packaging a 3′UTR deleted or otherwise “abbreviated” 3′ UTR GAA construct (asdiscussed in more detail below) into viral vectors (e.g., AAV vectors)can be improved. Further, the level of expression of the GAA mRNA and/orpolypeptide can be enhanced (e.g., as a result of a higher level oftranscription and/or translation and/or longer half-life of the mRNAtranscript and/or polypeptide) as compared with a full-length construct(e.g., SEQ ID NO:1 FIG. 8).

The present invention provides isolated nucleic acids encoding GA,comprising a coding sequence for GAA and an “abbreviated” 3′untranslated region (UTR). The “abbreviated” 3′ UTR are altered ascompared with the native GAA 3′ UTR sequence (e.g., the 3′ UTR of SEQ IDNO:1 is from nt 3301 through 3846; see also FIG. 8). In representativeembodiments, the abbreviated 3′ UTR comprises, consists essentially ofor consists of a deleted GAA 3′ UTR. In other embodiments, theabbreviated 3′ UTR is shortened as compared with the native GAA 3′ UTRand has been altered to contain a region from a heterologous 3′ UTR,which can be a partially or wholly synthetic 3′ UTR sequence. Theseembodiments of the invention are discussed in more detail below. Theisolated nucleic acids encoding GAA and comprising an abbreviated 3′ UTRof the invention can provide for higher levels of GAA polypeptideexpression. In particular embodiments, the abbreviated constructs canalso be more efficiently packaged into viral vectors (e.g., rAAVvectors).

The abbreviated 3′ UTR of the invention encode functional 3′ UTR that,in the presence of all other necessary regulatory elements, permit theexpression of a functional GAA polypeptide from a GAA coding sequenceoperably associated therewith. In illustrative embodiments, the isolatednucleic acid comprising a coding sequence for GAA and an abbreviated 3′UTR further comprises a secretory signal sequence operably linked to theGAA coding sequence, as described hereinabove.

While not wishing to be held to any particular theory of the invention,the improved properties of “abbreviated” 3′ UTR nucleic acids encodingGAA may be a result of their shorter total size and/or removal of aninhibitory region, e.g., a region that reduces transcription,destabilizes the mRNA transcript and/or inhibits translation. Examplesof small sequences that destabilize mRNA for cytokines (see, e.g., Shawand Kamen, (1986) Cell 46:659-67; Reeves et al., (1987) Proc. Natl.Acad. Sci. USA 84:6531-35) and the HIV gag gene (see. e.g., Schwartz etal., (1992) J. Virology 66:150-59; Schwartz et al., (1992) J. Virol.66:7176-82) have been described.

In particular embodiments, the isolated nucleic acid comprising the GAAcoding sequence and abbreviated 3′ UTR is less than about 4.5, 4.4, 4.3,4.2, 4.1, 4, 3.9 or 3.8 kb in length. To illustrate, according torepresentative embodiments, a GAA expression construct including 5′ and3′ UTR sequences is less than about 4.5, 4.4, 4.3, 4.2, 4.1, 4, 3.9 or3.8 kb in length.

The isolated nucleic acid encoding GAA can further comprise a 5′ UTR,which can further include all or a portion of the 5′ UTR of a GAA gene.Human GAA sequences with deletions in the 5′ UTR have been described,see, e.g., Van Hove et al. (1996) Proc. Natl. Acad. Sci. USA 93:65, inwhich nt 1-409 of the 5′ UTR of SEQ ID NO:1 (FIG. 8) have been deleted(see also SEQ ID NO:3; FIG. 9). Alternatively, the 5′ UTR can be derivedin whole or in part from a heterologous gene (i.e., a gene other than aGAA gene) and/or can comprise in whole or in part synthetic sequences.

As one aspect, the present invention provides isolated nucleic acidsthat encode GAA, where the isolated nucleic acid comprises (i) a GAAcoding sequence encoding a GAA and (ii) a GAA 3′ UTR region having adeletion therein.

A “coding region encoding a GAA polypeptide” comprises nucleotidesequences that can be transcribed and translated to yield a functionalGAA polypeptide or functional fragment thereof (see above). Such codingsequences may include non-translated sequences (e.g., intron sequences).

A “GAA 3′ UTR region” refers to the non-translated nucleic acidsequences of a GAA gene that are located downstream of (i.e., 3′ to) theregions of the gene that encode the GAA protein.

By “deleted” GAA 3′ UTR, it is intended that there is an omission of atleast one nucleotide from the 3′ UTR region of the GAA expressionconstruct. Deletions can be greater than about 25, 50, 100, 150, 200,300, 400 consecutive nucleotides, or more. In particular embodiments,essentially all of the 3′ UTR is deleted. By “essentially all” it ismeant that only an insignificant fragment of the intact 3′ UTR remains(i.e., less than 10, 20 or 30 nucleotides). For example, essentiallyall, but not necessarily all, of the 3′ UTR can be conveniently removedusing restriction enzymes (i.e., there may be some residual nucleotidesleft after restriction enzyme cleavage) or some untranslated nucleotidesmay remain 3′ of the coding sequence as an artifact of cloningprocedures.

Alternatively stated, at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, 99% ormore of the GAA 3′ UTR can be deleted. As still a further alternative,in embodiments of the invention, the “deleted” GAA 3′ UTR is less thanabout 300, 250, 200, 175, 150, 125, 100, 75, 50, 30, 20 or 10nucleotides in length or less.

In particular embodiments of the invention, the deleted 3′ UTRcomprises, consists essentially of or consists of a deleted form of the3′ UTR in the human GAA sequence provided in SEQ ID NO:1 (i.e., the 3′UTR of SEQ ID NO:1 is from nt 3301 through 3846; see also FIG. 8). Forexample, the deleted 3′ UTR can comprise, consist essentially of orconsist of the 3′ UTR shown in SEQ ID NO:3 (i.e., the 3′ UTR of SEQ IDNO:3 is from nt 2878 through 3012; see also, FIG. 9).

Referring to the 3′ UTR in the GAA sequence of SEQ ID NO:1 and FIG. 8(nt 3301 through 3846), the deleted 3′ UTR can comprise a deletion fromnt 3301 through 3846 of SEQ ID NO:1. The deletion can also encompassfrom about nt 3400 through nt 3500, nt 3500 through nt 3600, nt 3600through nt 3700, nt 3700 through nt 3800, nt 3800 through nt3846, nt3301 through nt 3450, nt 3450 through nt 3600, nt 3600 through nt 3750,nt 3750 through nt 3846, nt 3301 through nt 3500, nt 3400 through nt3600, nt 3500 through nt 3700, nt 3600 through nt 3800, nt 3700 throughnt 3846, nt 3301 through nt 3600, nt 3400 through nt 3700, nt 3500through nt 3800, nt 3600 through nt 3846, nt 3301 through nt 3700, nt3400 through nt 3800, nt 3500 through nt 3845, nt 3300 through nt 3800,or nt 3400 through nt 3486.

Deletions can be intermittent, i.e., more than one region of thenucleotide sequence can be deleted to impart the functional improvementsdescribed herein. Alternatively, consecutive nucleotides can be deleted.Further, the deletion can be internal or start at either end of the 3′UTR. For example, the 3′ UTR sequence can be truncated from the 5′ or 3′end of the 3′ UTR by 50, 100, 200, 300 or 400 nt or more.

Those skilled in the art will readily appreciate that the deleted andintact (i.e., from which the deleted 3′ UTR are derived) GAA 3′ UTRregions within the scope of the present invention can deviate from thosespecifically disclosed herein and that any suitable GAA coding sequenceor GAA 3′ UTR may be employed. The GAA coding sequences and 3′ UTR cancontain other alterations such as substitutions or insertions therein.For example, It will be understood that the GAA 3′ UTR can contain someheterologous sequence(s) (e.g., the polyA signal may be from anothergene, such as the human or bovine growth hormone gene).

In embodiments of the invention, the 3′ UTR deleted nucleic acidencoding GAA will hybridize to the 3′ UTR deleted nucleic acid sequencesspecifically disclosed herein (i.e., SEQ ID NO:3) under standardconditions as known by those skilled in the art and encode a functionalGAA polypeptide (as defined above).

In other embodiments, the deleted GAA 3′ UTR of the invention willhybridize to the deleted GAA 3′ UTR sequences specifically disclosedherein (e.g., nt 2878 to 3012 of SEQ ID NO:3) under standard conditionsas known by those skilled in the art and permit the expression of afunctional GAA polypeptide from a GAA coding sequence operablyassociated therewith.

In still further embodiments, the coding sequence of the isolatednucleic acid encoding GAA will hybridize to the sequences encoding GAAspecifically disclosed herein (e.g., nt 442 to nt 3300 of SEQ ID NO:1)under standard conditions as known by those skilled in the art andencode a functional GAA polypeptide.

For example, hybridization of such sequences can be carried out underconditions of reduced stringency, medium stringency or even stringentconditions (e.g., conditions represented by a wash stringency of 35-40%formamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at 37° C.;conditions represented by a wash stringency of 40-45% formamide with5×Denhardt's solution 0.5% SDS, and 1×SSPE at 42° C. and conditionsrepresented by a wash stringency of 50% formamide with 5×Denhardt'ssolution, 0.5% SDS and 1×SSPE at 42° C. respectively) to the sequencesspecifically disclosed herein. See, e.g., Sambrook et al., MolecularCloning, A Laboratory Manual (2d Ed. 1989) (Cold Spring HarborLaboratory).

Alternatively stated, in embodiments of the invention, 3′ UTR deletednucleic acid encoding GAA of the invention have at least about 60%, 70%,80%, 90%, 95%, 97%, 98% or higher nucleotide sequence homology with theisolated nucleic acid sequences specifically disclosed herein (orfragments thereof) and encode a functional GAA protein (mature orprecursor forms).

Likewise, in embodiments of the invention, the deleted 3′ UTR accordingto the present invention have at least about 60%, 70%, 80%, 90%, 95%,97%, 98%, or higher nucleotide sequence homology with the isolatednucleic acid sequences specifically disclosed herein (or fragmentsthereof) and permit the expression of a functional GAA polypeptide froma GAA coding sequence operably associated therewith.

Further, in embodiments of the invention, the coding region of theisolated nucleic acids encoding GAA of the invention have at least about60%, 70%, 80%, 90%, 95%, 97%, 98% or higher nucleotide sequence homologywith the isolated nucleic acid sequences specifically disclosed herein(or fragments thereof) and encode a functional GAA polypeptide.

It will be appreciated by those skilled in the art that there may bevariability in the polynucleotides that encode the GAA proteins of thepresent invention due to the degeneracy of the genetic code. Thedegeneracy of the genetic code, which allows different nucleic acidsequences to code for the same polypeptide, is well known in theliterature (see Table 1).

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCT Cysteine Cys CTGC TGT Aspartic acid Asp D GAC GAT Glutamic acid Glu E GAA GAGPhenylalanine Phe F TTC TTT Glycine Gly G GGA GGC GGG GGT Histidine HisH CAC CAT Isoleucine Ile I ATA ATC ATT Lysine Lys K AAA AAG Leucine LeuL TTA TTG CTA CTC CTG CTT Methionine Met M ATG Asparagine Asn N AAC AATProline Pro P CCA CCC CCG CCT Glutamine Gln Q CAA CAG Arginine Arg RAGA AGG CGA CGC CGG CGT Serine Ser S AGC ACT TCA TCC TCG TCT ThreonineThr T ACA ACC ACG ACT Valine Val V GTA GTC GTG GTT Tryptophan Trp W TGGTyrosine Tyr Y TAC TAT

Further, in other embodiments, the isolated nucleic acids of theinvention encompass those nucleic acids encoding GAA polypeptides thathave at least about 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or higheramino acid sequence homology with the polypeptide sequences specificallydisclosed herein (or fragments thereof) and encode a functional GAApolypeptide.

As is known in the art, a number of different programs can be used toidentify whether a nucleic acid or polypeptide has sequence identity orsimilarity to a known sequence. Sequence identity and/or similarity canbe determined using standard techniques known in the art, including, butnot limited to, the local sequence identity algorithm of Smith &Waterman, Adv. Appl. Math. 2, 482 (1981), by the sequence identityalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48, 443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Natl.Acad. Sci. USA 85, 2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Drive, Madison,Wis.), the Best Fit sequence program described by Devereux et al., Nucl.Acid Res. 12, 387-395 (1984), preferably using the default settings, orby inspection.

An example of a useful algorithm is PILEUP, which creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, J. Mol. Evol.35, 351-360 (1987); the method is similar to that described by Higgins &Sharp, CABIOS 5, 151-153 (1989).

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin etal., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993). A particularlyuseful BLAST program is the WU-BLAST-2 program which was obtained fromAltschul et al., Methods in Enzymology, 266, 460-480 (1996);http://blast.wustl/edu/blast/README.html. WU-BLAST-2 uses several searchparameters, which are preferably set to the default values. Theparameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity.

An additional useful algorithm is capped BLAST as reported by Altschulet al. Nucleic Acids Res. 25, 3389-3402.

A percentage amino acid sequence identity value can be determined by thenumber of matching identical residues divided by the total number ofresidues of the “longer” sequence in the aligned region. The “longer”sequence is the one having the most actual residues in the alignedregion (gaps introduced by WU-Blast-2 to maximize the alignment scoreare ignored).

The alignment can include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than the polypeptides specifically disclosed herein,it is understood that in one embodiment, the percentage of sequenceidentity will be determined based on the number of identical amino acidsin relation to the total number of amino acids. Thus, for example,sequence identity of sequences shorter than a sequence specificallydisclosed herein, will be determined using the number of amino acids inthe shorter sequence, in one embodiment. In percent identitycalculations relative weight is not assigned to various manifestationsof sequence variation, such as, insertions, deletions, substitutions,etc.

In one embodiment, only identities are scored positively (+1) and allforms of sequence variation including gaps are assigned a value of “0,”which obviates the need for a weighted scale or parameters as describedbelow for sequence similarity calculations. Percent sequence identitycan be calculated, for example, by dividing the number of matchingidentical residues by the total number of residues of the “shorter”sequence in the aligned region and multiplying by 100. The “longer”sequence is the one having the most actual residues in the alignedregion.

In a further representative embodiment of the invention, the isolatednucleic acid encoding GAA comprises an abbreviated 3′ UTR that isshorter than the 3′ UTR found in the native gene (e.g., nt 3301 to nt3846 of SEQ ID NO:1), to illustrate, is less than about 75%, 50%, 40%,30%, 20%, 10%, 5%, 4%, or less, of the size of the 3′ UTR found in thenative GAA gene, and comprises a heterologous region that is substitutedfor all or a portion of the native 3′ UTR. According to this embodiment,all or at least a portion of the 3′ UTR is heterologous to the GAAcoding region (i.e., is not derived from the 3′ UTR of a GAA gene). Theheterologous segment can include all or a portion of a 3′ UTR of anothergene and/or can be partially or completely synthetic. In particularembodiments, the heterologous region can be about 300, 250, 200, 1.75,125, 100, 75, 50, 30, 20 or 10 nucleotides in length or less. Toillustrate, the substituted 3′ UTR can include all or a portion of thebovine or human growth hormone 3′ UTR.

According to some embodiments of the invention, the total size of theabbreviated 3′ UTR is less than about 300, 250, 200, 175, 150, 125, 100,75, 50, 30, 20 or 10 nucleotides.

By “substitute,” “substituted” or “substitution” in reference to the 3′UTR is meant that a portion of the naturally-occurring nucleotidesequence of the GAA 3′ UTR has been replaced by a heterologousnucleotide sequence, resulting in a nucleic acid encoding GAA having theadvantages described herein.

According to the present invention, an “abbreviated” 3′ UTR encompassesboth deleted GAA 3′ UTR (described at length above) and thesubstituted/shortened 3′ UTR described in the preceding paragraphs. Theabbreviated 3′ UTR of the invention may be DNA or RNA, or a chimerathereof.

It still other embodiments of the invention, the “abbreviated” 3′ UTR isless than about 300, 250, 200, 150 or 100 nucleotides in length and isderived in whole or in part from a native GAA 3′ UTR and/or in whole orin part from a heterologous 3′ UTR. Further, the heterologous 3′ UTRsequences can be derived from another gene or can be in whole or in parta synthetic sequence.

As described in more detail below, the abbreviated 3′ UTR nucleic acidsof the invention can, upon introduction into a target cell (e.g., aliver cell), express GAA polypeptide at an enhanced (as defined above)level as compared with a cell expressing GAA polypeptide from acomparable construct that contains a full-length GAA 3′ UTR (e.g., SEQID NO:1).

III. Nucleic Acid Delivery Vectors

The methods of the present invention provide a means for delivering and,optionally, expressing lysosomal polypeptides such as GAA in a broadrange of host cells, including both dividing and non-dividing cells invitro or in vivo. In embodiments of the invention, the nucleic acid maybe stably introduced into the target cell, for example, by integrationinto the genome of the cell or by persistent expression from stablymaintained episomes (e.g., derived from Epstein Barr Virus).Alternatively, the isolated nucleic acid can be transiently expressed inthe cell.

The isolated nucleic acids, vectors, cells, methods and pharmaceuticalformulations of the present invention are additionally useful in amethod of administering lysosomal polypeptides such as GAA to a subjectin need thereof. In this manner, the polypeptide can thus be produced invivo in the subject. The subject may have a deficiency of thepolypeptide, or the production of a foreign polypeptide in the subjectmay impart some therapeutic effect. Pharmaceutical formulations andmethods of delivering lysosomal polypeptides such as GAA for therapeuticpurposes are described in more detail in Section V below.

Alternatively, a polynucleotide encoding and expressing the lysosomalpolypeptide (e.g., GAA) can be administered to a subject so that thepolypeptide is expressed by the subject and purified therefrom, i.e., asa source of recombinant polypeptide. According to this embodiment, it ispreferred that the polypeptide is secreted into the systemic circulationor into another body fluid (e.g., milk, lymph, spinal fluid, urine) thatis easily collected and from which the polypeptide can be furtherpurified. Alternatively, the polypeptide can be expressed in avianspecies and deposited in, and conveniently isolated from, egg proteins.

Likewise, the polypeptide can be expressed transiently or stably in acell culture system. In particular embodiments, the polypeptide issecreted into the medium and can be purified therefrom using routinetechniques known in the art. Additionally. or alternatively the cellscan be lysed and the recombinant polypeptide can be purified from thecell lysate. The cell may be a bacterial, protozoan, plant, yeast,fungus, or animal cell. The cell can be an animal cell (e.g., insect,avian or mammalian). Representative mammalian cells include but are notlimited to fibroblasts, CHO cells, 293 cells, HT1080 cells. HeLa cellsand C10 cells.

In the case of GAA, the recombinant GAA polypeptide can be isolatedusing standard techniques and administered to subjects with GAAdeficiency using enzyme replacement protocols (see, e.g., Van der Ploeget al., (1991) J. Clin. Invest. 87:513).

Transfer of a nucleic acid encoding a lysosomal polypeptide (e.g., GAA)to a cell in culture or to a subject also finds use as a model forunderstanding disease states such as GSD II and for investigating thebiology of these polypeptides.

Still further, the instant invention finds use in screening methods,whereby the polypeptide is transiently or stably expressed in a cellculture system or animal model and used as a target for drug discovery.

Methods of producing lysosomal polypeptides such as GAA in culturedcells or organisms for the purposes described above are set forth inmore detail in Section IV below.

It will be apparent to those skilled in the art that any suitable vectormay be used to deliver the isolated nucleic acids of the invention tothe target cell(s) or subject of interest. The choice of delivery vectormay be made based on a number of factors known in the art, including ageand species of the target host, in vitro vs. in vivo delivery, level andpersistence of expression desired, intended purpose (e.g., for therapyor enzyme production), the target cell or organ, route of delivery, sizeof the isolated nucleic acid, safety concerns, and the like.

Any suitable vector known in the art can be used to deliver, andoptionally, express the isolated nucleic acids of the invention,including, virus vectors (e.g., retrovirus, adenovirus, adeno-associatedvirus, or herpes simplex virus), lipid vectors, poly-lysine vectors,synthetic polyamino polymer vectors that are used with nucleic acidmolecules, such as a plasmid, and the like.

Any viral vector that is known in the art may be used in the presentinvention. Examples of such viral vectors include, but are not limitedto vectors derived from: Adenoviridae; Bimavindae; Bunyaviridae;Calicivindae, Capillovirus group; Carlavirus group; Carmovirus virusgroup; Group Caulimovirus; Closterovirus Group; Commelina yellow mottlevirus group; Comovirus virus group; Coronaviridae; PM2 phage group;Corcicovtridae; Group Cryptic virus; group Cryptovirus; Cucumovirusvirus group family ([PHgr]6 phage group; Cysioviridae; Group Carnationringspot; Dianthovirus virus group; Group Broad bean wilt; Fabavirusvirus group; Filoviridae; Flaviviridae; Furovirus group; GroupGerminivirus; Group Giardiavirus; Hepadnaviridae; Herpesviridae;Hordeivirus virus group; Illarvirus virus group; inoviridae;Iridoviridae; Leviviridae; Lipothrixviridae; Luteovirus group;Marafivirus virus group; Maize chlorotic dwarf virus group; icroviridae;Myoviridae; Necrovirus group; Nepovirus virus group; Nodaviridae;Orthomyxoviridae; Papovaviridae; Paramyxoviridae; Parsnip yellow fleckvirus group; Partitiviridae; Parvoviridae; Pea enation mosaic virusgroup; Phycodnaviridae; Picomaviridae; Plasmaviridae; Prodovindae;Polydnaviridae; Potexvirus group; Potyvirus; Poxviridae; Reovindae;Retroviridae; Rhabdoviridae; Group Rhizidiovirus; Siphovindae;Sobemovirus group; SSV 1-Type Phages; Tectiviridae; Tenuivirus;Tetravindae; Group Tobamovirus; Group Tobravirus; Togaviridae; GroupTombusvirus; Group Torovirus; Totiviridae; Group Tymovirus; and plantvirus satellites.

Protocols for producing recombinant viral vectors and for using viralvectors for nucleic acid delivery can be found in Current Protocols inMolecular Biology, Ausubel, F. M. et al. (eds.) Greene PublishingAssociates, (1989) and other standard laboratory manuals (e.g., Vectorsfor Gene Therapy. In: Current Protocols in Human Genetics. John Wileyand Sons, Inc.: 1997).

Particularly preferred viral vectors are those previously employed forthe delivery of transgenes including, for example, retrovirus,adenovirus, AAV, herpes virus, hybrid adenovirus-AAV, and poxvirusvectors. In particular embodiments, the vector is an adenovirus vector,AAV vector or hybrid Ad-AAV vector.

In certain preferred embodiments of the present invention, the deliveryvector is an adenovirus vector. The term “adenovirus” as used herein isintended to encompass all adenoviruses, including the Mastadenovirus andAviadenovirus genera. To date, at least forty-seven human serotypes ofadenoviruses have been identified (see, e.g., FIELDS et al., VIROLOGY,volume 2, chapter 67 (3d ed., Lippincott-Raven Publishers)). Preferably,the adenovirus is a serogroup C adenovirus, still more preferably theadenovirus is serotype 2 (Ad2) or serotype 5 (Ad5).

The various regions of the adenovirus genome have been mapped and areunderstood by those skilled in the art (see, e.g., FIELDS et al.,VIROLOGY, volume 2, chapters 67 and 68 (3d ed., Lippincott-RavenPublishers)). The genomic sequences of the various Ad serotypes, as wellas the nucleotide sequence of the particular coding regions of the Adgenome, are known in the art and may be accessed, e.g., from GenBank andNCBI (see, e.g., GenBank Accession Nos. J0917, M73260, X73487, AF108105,L19443, NC 003266 and NCBI Accession Nos. NC 001405, NC 001460, NC002067, NC 00454).

Those skilled in the art will appreciate that the inventive adenovirusvectors may be modified or “targeted” as described in Douglas et al.,(1996) Nature Biotechnology 14:1574; U.S. Pat. No. 5,922,315 to Roy etal.; U.S. Pat. No. 5,770,442 to Wickham et al.; and/or U.S. Pat. No.5,712,136 to Wickham et al.

An adenovirus vector genome or rAd vector genome will typically comprisethe Ad terminal repeat sequences and packaging signal. An “adenovirusparticle” or “recombinant adenovirus particle” comprises an adenovirusvector genome or recombinant adenovirus vector genome, respectivelypackaged within an adenovirus capsid. Generally, the adenovirus vectorgenome is most stable at sizes of about 28 kb to 38 kb (approximately75% to 105% of the native genome size). In the case of an adenovirusvector containing large deletions and a relatively small transgene.“stuffer DNA” can be used to maintain the total size of the vectorwithin the desired range by methods known in the art.

Normally adenoviruses bind to a cell surface receptor (CAR) ofsusceptible cells via the knob domain of the fiber protein on the virussurface. The fiber knob receptor is a 45 kDa cell surface protein whichhas potential sites for both glycosylation and phosphorylation.(Bergelson, et al., (1997) Science 275:1320-1323. A secondary method ofentry for adenovirus is through integrins present on the cell surface.Arginine-Glycine-Aspartic Acid (RGD) sequences of the adenoviral pentonbase protein bind integrins on the cell surface.

The genome of an adenovirus can be manipulated such that it encodes andexpresses a gene product of interest but is inactivated in terms of itsability to replicate in a normal lytic viral life cycle. See, forexample, Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al.(1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.Suitable adenoviral vectors derived from the adenovirus strain Ad type 5dl 324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7, etc.) areknown to those skilled in the art. Recombinant adenoviruses can beadvantageous in certain circumstances in that they are not capable ofinfecting nondividing cells and can be used to infect a wide variety ofcell types, including epithelial cells. Furthermore, the virus particleis relatively stable and amenable to purification and concentration, andas above, can be modified so as to affect the spectrum of infectivity.Additionally, introduced adenoviral DNA (and foreign DNA containedtherein) is not integrated into the genome of a host cell but remainsepisomal, thereby avoiding potential problems that can occur as a resultof insertional mutagenesis in situations where introduced DNA becomesintegrated into the host genome (e.g., retroviral DNA). Moreover, thecarrying capacity of the adenoviral genome for foreign DNA is largerelative to other nucleic acid delivery vectors (Haj-Ahmand and Graham(1986) J. Virol. 57:267).

In particular embodiments, the adenovirus genome contains a deletiontherein, so that at least one of the adenovirus gene regions does notencode a functional protein. For example, first-generation adenovirusvectors are typically deleted for the E1 genes and packaged using a cellthat expresses the E1 proteins (e.g., 293 cells). The E3 region is alsofrequently deleted as well, as there is no need for complementation ofthis deletion. In addition, deletions in the E4, E2a, protein IX, andfiber protein regions have been described, e.g., by Armentano et al,(1997) J. Virology 71:2408, Gao et al., (1996) J. Virology 70:8934,Dedieu et al., (1997) J. Virology 71:4626, Wang et al., (1997) GeneTherapy 4:393, and U.S. Pat. No. 5,882,877 to Gregory et al. (thedisclosures of which are incorporated herein in their entirety).Preferably, the deletions are selected to avoid toxicity to thepackaging cell. Wang et al., (1997) Gene Therapy 4:393, has describedtoxicity from constitutive co-expression of the E4 and E1 genes by apackaging cell line. Toxicity may be avoided by regulating expression ofthe E1 and/or E4 gene products by an inducible, rather than aconstitutive, promoter. Combinations of deletions that avoid toxicity orother deleterious effects on the host cell can be routinely selected bythose skilled in the art.

As further examples, in particular embodiments, the adenovirus isdeleted in the polymerase (pol), preterminal protein (pTP), IVa2 and/or100K regions (see, e.g., U.S. Pat. No. 6,328,958; PCT publication WO00/12740; and PCT publication WO 02/098466; Ding et al., (2002) Mol.Ther. 5:436; Hodges et al., J. Virol. 75:5913; Ding et al., (2001) HumGene Ther 12:955; the disclosures of which are incorporated herein byreference in their entireties for the teachings of how to make and usedeleted adenovirus vectors for nucleic acid delivery). In representativeembodiments, the vector is a [E1−, E3−, pol−]Ad, [E1−, E3−, pTP−]Ad,[E1−, E3−, pol1, pTP−J]Ad, [E1+, 100K−]Ad or [E1a+, E1b−, 100K]Ad.

The term “deleted” adenovirus as used herein refers to the omission ofat least one nucleotide from the indicated region of the adenovirusgenome. Deletions can be greater than about 1, 2, 3, 5, 10, 20, 50, 100,200, or even 500 nucleotides. Deletions in the various regions of theadenovirus genome may be about at least 1%, 5%, 10%, 25%, 50%, 75%, 90%,95%, 99%, or more of the indicated region. Alternately, the entireregion of the adenovirus genome is deleted. Preferably, the deletionwill prevent or essentially prevent the expression of a functionalprotein from that region. For example, it is preferred that the deletionin the 100K region results in the loss of expression of a functional100K protein from that region. In other words, even if there istranscription across the deleted 100K region and translation of theresulting RNA transcripts, the resulting protein will be essentiallynon-functional, more preferably, completely non-functional.Alternatively, an insignificant amount of a functional protein isexpressed. In general, larger deletions are preferred as these have theadditional advantage that they will increase the carrying capacity ofthe deleted adenovirus for a heterologous nucleotide sequence ofinterest. The various regions of the adenovirus genome have been mappedand are understood by those skilled in the art (see, e.g., FIELDS etal., VIROLOGY, volume 2, chapters 67 and 68 (3d ed., Lippincott-RavenPublishers)).

Those skilled in the art will appreciate that typically, with theexception of the E3 genes, any deletions will need to be complemented inorder to propagate (replicate and package) additional virus, e.g., bytranscomplementation with a packaging cell.

In particular embodiments, the present invention excludes “guttedadenovirus” vectors (as that term is understood in the art, see e.g.,Lieber, et al., (1996) J. Virol. 70:8944-60) in which essentially all ofthe adenovirus genomic sequences are deleted. In alternate embodiments,such gutted adenovirus vectors may be an aspect of the invention.

Adeno-associated viruses (AAV) have also been employed as nucleic aciddelivery vectors. For a review, see Muzyczka et al. Curr Topics inMicro. and Immunol. (1992) 158:97-129). AAV are parvoviruses and havesmall icosahedral virions, 18-26 nanometers in diameter and contain asingle stranded DNA molecule 4-5 kilobases in size. The viruses containeither the sense or antisense strand of the DNA molecule and eitherstrand is incorporated into the virion. Two open reading frames encode aseries of Rep and Cap polypeptides. Rep polypeptides (Rep50, Rep52,Rep68 and Rep78) are involved in replication, rescue and integration ofthe AAV genome, although significant activity may be observed in theabsence of all four Rep polypeptides. The Cap proteins (VP1, VP2, VP3)form the virion capsid. Flanking the rep and cap open reading frames atthe 5′ and 3′ ends of the genome are 145 basepair inverted terminalrepeats (ITRs), the first 125 basepairs of which are capable of formingY- or T-shaped duplex structures. It has been shown that the ITRsrepresent the minimal cis sequences required for replication, rescue,packaging and integration of the AAV genome. Typically, in recombinantAAV vectors (rAAV), the entire rep and cap coding regions are excisedand replaced with a transgene of interest.

AAV are among the few viruses that may integrate their DNA intonon-dividing cells, and exhibit a high frequency of stable integrationinto human chromosome 19 (see, for example, Flotte et al. (1992) Am. J.Respir. Cell. Mol. Biol. 7:349-356; Samulski et al., (1989) J Virol.63:3822-3828; and McLaughlin et al., (1989) J. Virol. 62:1963-1973). Avariety of nucleic acids have been introduced into different cell typesusing AAV vectors (see, for example, Hermonat et al., (1984) Proc. Natl.Acad. Sci. USA 81:6466-6470; Tratschin et al., (1985) Mol. Cell. Biol.4:2072-2081; Wondisford et al., (1988) Mol. Endocrinol. 2:32-39;Tratschin et al., (1984) J. Virol. 51:611-619; and Flotte et al., (1993)J. Biol. Chem. 268:3781-3790).

A rAAV vector genome will typically comprise the AAV terminal repeatsequences and packaging signal. An “AAV particle” or “rAAV particle”comprises an AAV vector genome or rAAV vector genome, respectively,packaged within an AAV capsid. The rAAV vector itself need not containAAV genes encoding the capsid and Rep proteins. In particularembodiments of the invention, the rep and/or cap genes are deleted fromthe AAV genome. In a representative embodiment, the rAAV vector retainsonly the terminal AAV sequences (ITRs) necessary for integration,excision, replication.

Sources for the AAV capsid genes may include serotypes AAV-1, AAV-2,AAV-3 (including 3a and 3b). AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, as wellas bovine AAV and avian AAV, and any other virus classified by theinternational Committee on Taxonomy of Viruses (ICTV) as an AAV (see.e.g., BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed.,Lippincott-Raven Publishers)).

In particular embodiments, the AAV capsid genes are derived from AAVserotypes 1, 2, 5, 6 or 8.

Because of packaging limitations, the total size of the rAAV genome willpreferably be less than about 5.2, 5, 4.8, 4.6 or 4.5 kb in size.

Any suitable method known in the art may be used to produce AAV vectors(see, e.g., U.S. Pat. No. 5,139,941; U.S. Pat. No. 5,858,775; U.S. Pat.No. 6,146,874 for illustrative methods). In one particular method, AAVstocks may be produced by co-transfection of a rep/cap vector encodingAAV packaging functions and the template encoding the AAV vDNA intohuman cells infected with the helper adenovirus (Samulski et al., (1989)J. Virology 63:3822).

In other particular embodiments, the adenovirus helper virus is a hybridhelper virus that encodes AAV Rep and/or capsid proteins. Hybrid helperAd/AAV vectors expressing AAV rep and/or cap genes and methods ofproducing AAV stocks using these reagents are known in the art (see,e.g., U.S. Pat. No. 5,589,377; and U.S. Pat. No. 5,871,982, U.S. Pat.No. 6,251,677; and U.S. Pat. No. 6,387,368). Preferably, the hybrid Adof the invention expresses the AAV capsid proteins (i.e., VP1, VP2, andVP3). Alternatively, or additionally, the hybrid adenovirus may expressone or more of AAV Rep proteins (i.e., Rep40, Rep52, Rep68 and/orRep78). The AAV sequences may be operatively associated with atissue-specific or inducible promoter.

The AAV rep and/or cap genes may alternatively be provided by apackaging cell that stably expresses the genes (see, e.g., Gao et al.,(1998) Human Gene Therapy 9:2353; Inoue et al., (1998) J. Virol.72:7024; U.S. Pat. No. 5,837,484; WO 98/27207; U.S. Pat. No. 5,658,785;WO 96/17947).

In a representative embodiment, the present invention provides a methodof producing a rAAV particle comprising an isolated nucleic acidaccording to the invention, comprising providing to a cell: (a) anucleic acid encoding a rAAV genome comprising (i) 5′ and/or 3′ AAV ITRsequences. (ii) an isolated nucleic acid as described above (e.g., anucleic acid encoding GAA and comprising an abbreviated 3′ UTR or anucleic acid encoding a chimeric lysosomal polypeptide comprising asecretory signal sequence), and (iii) an AAV packaging signal; (b) AAVrep coding sequences sufficient for replication of the recombinant AAVgenome; (c) AAV cap coding sequences sufficient to produce a functionalAAV capsid; wherein (a) to (c) are provided to the cell under conditionssufficient for replication and packaging of the rAAV genome into the AAVcapsid, whereby AAV particles comprising the AAV capsid packaging therAAV genome are produced in the cell. Typically, the adenovirus or HSVhelper functions for AAV replication and packaging are also provided.The method may further include the step of collecting the rAAVparticles.

In still further embodiments, the delivery vector is a hybrid. Ad-AAVdelivery vector, for example, as described in the working Examples andin U.S. Provisional Application 60/376,397 (incorporated by referenceherein in its entirety for its teaching of how to make and use hybridAd-AAV delivery vectors). Briefly, the hybrid Ad-AAV vector comprises anadenovirus vector genome comprising adenovirus (i) 5′ and 3′cis-elements for viral replication and encapsidation and, further, (ii)a recombinant AAV vector genome comprising the AAV 5′ and 3′ invertedterminal repeats (ITRs), an AAV packaging sequence, and a heterologoussequence(s) flanked by the AAV ITRs, where the recombinant AAV vectorgenome is flanked by the adenovirus 5′ and 3′ cis-elements. Theadenovirus vector genome may further be deleted, as described above.

Another vector for use in the present invention comprises Herpes SimplexVirus (HSV). Herpes simplex virions have an overall diameter of 150 to200 nm and a genome consisting of one double-stranded DNA molecule thatis 120 to 200 kilobases in length. Glycoprotein D (gD) is a structuralcomponent of the HSV envelope that mediates virus entry into host cells.The initial interaction of HSV with cell surface heparin sulfateproteoglycans is mediated by another glycoprotein, glycoprotein C (gC)and/or glycoprotein B (gB). This is followed by interaction with one ormore of the viral glycoproteins with cellular receptors. Recently it hasbeen shown that glycoprotein D of HSV binds directly to Herpes virusentry mediator (HVEM) of host cells. HVEM is a member of the tumornecrosis factor receptor superfamily (Whitbeck. J. C. et al., 1997, J.Virol.; 71:6083-6093). Finally, gD, gB and the complex of gH and gL actindividually or in combination to trigger pH-independent fusion of theviral envelope with the host cell plasma membrane. The virus itself istransmitted by direct contact and replicates in the skin or mucosalmembranes before infecting cells of the nervous system for which HSV hasparticular tropism. It exhibits both a lytic and a latent function. Thelytic cycle results in viral replication and cell death. The latentfunction allows for the virus to be maintained in the host for anextremely long period of time.

HSV can be modified for the delivery of transgenes to cells by producinga vector that exhibits only the latent function for long-term genemaintenance. HSV vectors are useful for nucleic acid delivery becausethey allow for a large DNA insert of up to or greater than 20 kilobases;they can be produced with extremely high titers; and they have beenshown to express transgenes for a long period of time in the centralnervous system as long as the lytic cycle does not occur.

In other preferred embodiments of the present invention, the deliveryvector of interest is a retrovirus. Retroviruses normally bind to aspecies specific cell surface receptor, e.g., CD4 (for HIV); CAT (forMLV-E; ecotropic Murine leukemic virus E); RAM1/GLVR2 (for murineleukemic virus-A; MLV-A); GLVR1 (for Gibbon Ape leukemia virus (GALV)and Feline leukemia virus B (FeLV-B)). The development of specializedcell lines (termed “packaging cells”) which produce onlyreplication-defective retroviruses has increased the utility ofretroviruses for gene therapy, and defective retroviruses arecharacterized for use in gene transfer for gene therapy purposes (for areview, see Miller, (1990) Blood 76.271). A replication-defectiveretrovirus can be packaged into virions which can be used to infect atarget cell through the use of a helper virus by standard techniques.

Yet another suitable vector is a poxvirus vector. These viruses are verycomplex, containing more than 100 proteins, although the detailedstructure of the virus is presently unknown. Extracellular forms of thevirus have two membranes while intracellular particles only have aninner membrane. The outer surface of the virus is made up of lipids andproteins that surround the biconcave core. Poxviruses are very complexantigenically, inducing both specific and cross-reacting antibodiesafter infection. Poxvirus receptors are not presently known, but it islikely that there exists more than one given the ability of poxvirus toinfect a wide range of cells. Poxvirus gene expression is well studieddue to the interest in using vaccinia virus as a vector for expressionof transgenes.

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed. Many non-viral methods of genetransfer rely on normal mechanisms used by mammalian cells for theuptake and intracellular transport of macromolecules. In particularembodiments, non-viral delivery systems rely on endocytic pathways forthe uptake of the nucleic acid molecule by the targeted cell. Exemplarynucleic acid delivery systems of this type include liposomal derivedsystems, poly-lysine conjugates, and artificial viral envelopes.

In particular embodiments, plasmid vectors are used in the practice ofthe present invention. Naked plasmids can be introduced into musclecells by injection into the tissue. Expression can extend over manymonths, although the number of positive cells is typically low (Wolff etal., (1989) Science 247:247). Cationic lipids have been demonstrated toaid in introduction of DNA into some cells in culture (Felgner andRingold, (1989) Nature 337:387). Injection of cationic lipid plasmid DNAcomplexes into the circulation of mice has been shown to result inexpression of the DNA in lung (Brigham et al., (1989) Am. J. Med. Sci.298:278). One advantage of plasmid DNA is that it may be introduced intonon-replicating cells.

In a representative embodiment, a nucleic acid molecule (e.g., aplasmid) may be entrapped in a lipid particle bearing positive chargeson its surface and, optionally tagged with antibodies against cellsurface antigens of the target tissue (Mizuno et al., (1992) No ShinkeiGeka 20:547; PCT publication WO 91/06309; Japanese patent application1047381; and European patent publication EP-A-43075).

Liposomes that consist of amphiphilic cationic molecules are usefulnon-viral vectors for nucleic acid delivery in vitro and in vivo(reviewed in Crystal, Science 270: 404-410 (1995); Blaese et al., CancerGene Ther. 2: 291-297 (1995); Behr et al., Bioconjugate Chem. 5: 382-389(1994); Remy et al., Bioconjugate Chem. 5: 647-654 (1994); and Gao etal., Gene Therapy 2: 710-722 (1995)). The positively charged liposomesare believed to complex with negatively charged nucleic acids viaelectrostatic interactions to form lipid:nucleic acid complexes. Thelipid:nucleic acid complexes have several advantages as gene transfervectors. Unlike viral vectors, the lipid:nucleic acid complexes can beused to transfer expression cassettes of essentially unlimited size.Since the complexes lack proteins, they may evoke fewer immunogenic andinflammatory responses. Moreover, they cannot replicate or recombine toform an infectious agent and have low integration frequency. A number ofpublications have demonstrated that amphiphilic cationic lipids canmediate nucleic acid delivery in vivo and in vitro (Felgner et al.,Proc. Natl. Acad. Sci. USA 84: 7413-17 (1987); Loeffler et al., Methodsin Enzymology 217: 599-618 (1993); Felgner et al., J. Biol. Chem. 269:2550-2561 (1994)).

Several groups have reported the use of amphiphilic cationiclipid:nucleic acid complexes for in vivo transfection both in animalsand in humans (reviewed in Gao et al., Gene Therapy 2: 710-722 (1995);Zhu et al., Science 261: 209-211 (1993); and Thierry et al., Proc. Natl.Acad. Sci. USA 92: 9742-9746 (1995)). U.S. Pat. No. 6,410,049 describesa method of preparing cationic lipid:nucleic acid complexes that haveprolonged shelf life.

IV. Production of Recombinant Lysosomal Polypeptides.

As indicated above, recombinant lysosomal polypeptides such as GAA canbe produced in, and optionally purified from, cultured cells ororganisms for a variety of purposes. Methods of delivering a recombinantnucleic acid encoding a lysosomal polypeptide for therapeutic methodsare described in more detail below. The isolated nucleic acid may becarried by a delivery vector as described in the preceding section.

Those skilled in the art will appreciate that the isolated nucleic acidencoding the lysosomal polypeptide can be operably associated withappropriate expression control sequences, e.g.,transcription/translation control signals, which can be included in theisolated nucleic acid or by a vector backbone. For example, specificinitiation signals are generally required for efficient translation ofinserted protein coding sequences. These exogenous translational controlsequences, which may include the ATG initiation codon and adjacentsequences, can be of a variety of origins, both natural and synthetic.

The isolated nucleic acid can further comprise a polyadenylation signal(e.g., a signal for polyA polymerase to add the polyA tail to the 3′ endof the transcribed mRNA). It is common, however, that thepolyadenylation signal is provided by a vector backbone into which thecoding sequence is inserted (e.g., a plasmid or a recombinant viralgenome) and, therefore, in particular embodiments a polyadenylationsignal may not be present in the isolated nucleic acid molecule.

A variety of promoter/enhancer elements may be used depending on thelevel and tissue-specific expression desired. The promoter can beconstitutive or inducible (e.g., the metalothionein promoter or ahormone inducible promoter), depending on the pattern of expressiondesired. The promoter may be native or foreign and can be a natural or asynthetic sequence. By foreign, it is intended that the transcriptionalinitiation region is not found in the wild-type host into which thetranscriptional initiation region is introduced. The promoter is chosenso that it will function in the target cell(s) of interest. Promotersthat function in liver (for example, liver parenchyma, e.g., alpha-1antitrypsin promoter), skeletal muscle, cardiac muscle, smooth muscle,diaphragm muscle, endothelial cells, intestinal cells, pulmonary cells(e.g., smooth muscle or epithelium), peritoneal epithelial cells andfibroblasts are preferred. The promoter may further be “specific” forthese cells and tissues, in that it may only show significant activityin the specific cell or tissue type.

The isolated nucleic acid can be operatively associated with acytomegalovirus (CMV) major immediate-early promoter, an albuminpromoter, an Elongation Factor 1-α (EF1-α) promoter, a PγK promoter, aMFG promoter, or a Rous sarcoma virus promoter. A hybrid promotercontaining the CMV major immediate-early enhancer and chicken beta-actin(CB) promoter is also suitable. It has been speculated that drivingheterologous nucleotide transcription with the CMV promoter results indown-regulation of expression in immunocompetent animals (see, e.g., Guoet al., (1996) Gene Therapy 3:802). Accordingly, it may be advantageousto operably associate the isolated nucleic acid with a modified CMVpromoter that does not result in this down-regulation of transgeneexpression.

The isolated nucleic acids of the invention can comprise two or morecoding sequences. In embodiments wherein there is more than one codingsequence, the coding sequences may be operatively associated withseparate promoters or, alternatively, with a single upstream promoterand one or more downstream internal ribosome entry site (IRES) sequences(e.g., the picomavirus EMC IRES sequence).

In particular embodiments of the invention, the total size of theisolated nucleic acid is less than about 5, 4.8, 4.7, 4.6, 4.5, 4.3,4.2, 4, 3.8, 3.7, 3.6, 3.5, 3.2, 3 or 2.8 kb or less in length.Relatively small expression cassettes can be particularly advantageousfor delivery by AAV vectors.

An isolated nucleic acid of the invention can be introduced into a hostcell. e.g., a cell of a primary or immortalized cell line. Therecombinant cells can be used to produce the encoded polypeptide.Generally, the isolated nucleic acid is incorporated into an expressionvector (viral or nonviral as described above).

Expression vectors can be designed for expression of polypeptides inprokaryotic or eukaryotic cells. For example, polypeptides can beexpressed in bacterial cells such as E. coli, insect cells (e.g., in thebaculovirus expression system) yeast cells or mammalian cells. Somesuitable host cells are discussed further in Goeddel. Gene ExpressionTechnology: Methods in Enzymology 185. Academic Press, San Diego. Calif.(1990). Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSecl (Baldari et al., (1987) EMBO J. 6:229-234), pMFa(Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al.,(1987) Gene 54; 113-123), and pYES2 (Invitrogen Corporation, San Diego,Calif.). Baculovirus vectors available for expression of proteins incultured insect cells (e.g., Sf 9 cells) include the pAc series (Smithet al., (1983) Mol. Cell. Biol. 3:2156-2165) and the pVL series(Lucklow. V. A., and Summers, M. d. (1989) Virology 170:31-39).

Examples of mammalian expression vectors include pCDM8 (Seed, (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40.

In addition to the regulatory control sequences discussed above, therecombinant expression vector can contain additional nucleotidesequences. For example, the recombinant expression vector may encode aselectable marker gene to identify host cells that have incorporated thevector.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional technique including but not limited to calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, electroporation, microinjection and viral-mediatedtransfection and transduction. Suitable methods for transforming ortransfecting host cells can be found in Sambrook et al. (MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratorypress (1989)), and other laboratory manuals.

Often only a small fraction of cells (in particular, mammalian cells)integrate the foreign DNA into their genome. In order to identify andselect these integrants, a nucleic acid sequence that encodes aselectable marker (e.g., resistance to antibiotics) car be introducedinto the host cells along with the gene encoding the protein ofinterest. Preferred selectable markers include those that conferresistance to drugs, such as G418, hygromycin and methotrexate. Nucleicacids encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding the protein of interest or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

A. Transgenic Animals.

The lysosomal polypeptide may be produced in a non-human transgenicanimal (i.e., containing a nucleic acid introduced by human interventionusing recombinant nucleic acid techniques). Methods for generatingnon-human transgenic animals are known in the art. DNA constructs can beintroduced into the germ line of an avian or mammal to make a transgenicanimal. For example, one or several copies of the construct can beincorporated into the genome of an embryo by standard transgenictechniques.

It is often desirable to express the transgenic polypeptide in the milkof a transgenic mammal. Mammals that produce large volumes of milk andhave long lactating periods are preferred. Preferred mammals areruminants, e.g., cows, sheep, camels or goats (including goats of Swissorigin, such as the Alpine, Saanen and Toggenburg breed goats). Otherpreferred mammals include oxen, rabbits and pigs.

In an exemplary embodiment, a transgenic non-human animal is produced byintroducing a transgene into the germ line of the non-human animal.Transgenes can be introduced into embryonal target cells at variousdevelopmental stages. Different methods are used depending on the stageof development of the embryonal target cell. The specific line(s) of anyanimal used should, if possible, be selected for general good health,good embryo yields, good pronuclear visibility in the embryo, and goodreproductive fitness.

Introduction of the transgene into the embryo can be accomplished by anyof a variety of means known in the art such as microinjection,electroporation, lipofection or a viral vector. For example, thetransgene can be introduced into a mammal by microinjection of theconstruct into the pronuclei of the fertilized mammalian egg(s) to causeone or more copies of the construct to be retained in the cells of thedeveloping mammal(s). Following introduction of the transgene constructinto the fertilized egg, the egg can be incubated in vitro for varyingamounts of time, or reimplanted into the surrogate host, or both. Onecommon method is to incubate the embryos in vitro for about 1-7 days,depending on the species, and then reimplant them into the surrogatehost.

The progeny of the transgenically manipulated embryos can be tested forthe presence of the construct, e.g., by Southern blot analysis of asegment of tissue. An embryo having one or more copies of the exogenouscloned construct stably integrated into the genome can be used toestablish a permanent transgenic animal line carrying the transgenicconstruct.

Litters of transgenically altered mammals can be assayed after birth forthe incorporation of the construct into the genome of the offspring.This can be done by hybridizing a probe corresponding to the transgenicsequence to chromosomal material from the progeny. Those mammalianprogeny found to contain at least one copy of the construct in theirgenome are grown to maturity. The female species of these progeny willproduce the desired polypeptide in or along with their milk. Thetransgenic mammals can be bred to produce other transgenic progenyuseful in producing the desired polypeptides in their milk.

Transgenic females may be tested for polypeptide secretion into milk,using any art-known assay technique, e.g., a Western blot or enzymaticassay.

Useful transcriptional promoters for expressing the polypeptide in themilk of a transgenic animal are those promoters that are preferentiallyactivated in mammary epithelial cells, including promoters that controlthe genes encoding milk polypeptides such as caseins, beta-lactoglobulin(Clark et al., (989. Bio/Technology 7.487-492), whey acid protein(Gorton et a. (1987) Bio/Technology 5.1183-1187), and lactalbumin(Soulier et al., (1992) FEBS Letts. 297:13). The alpha-, beta-, gamma-or kappa-casein gene promoters of any mammalian species can be used toprovide mammary expression; a preferred promoter is the goat beta-caseingene promoter (DiTullio, (1992) Bio/Technology 10:74-77). Othermilk-specific polypeptide promoter or promoters that are specificallyactivated in mammary tissue can be isolated from cDNA or genomicsequences.

DNA sequence information is available for mammary gland specific geneslisted above, in at least one, and often in several organisms. See,e.g., Richards et al., J Biol. Chem. 256, 526-532 (1981) (ratalpha-lactalbumin); Campbell et al., Nucleic Acids Res. 12, 8685-8697(1984) (rat WAP); Jones et al., J. Biol. Chem. 260: 7042-7050 (1985)(rat beta-casein); Yu-Lee & Rosen, J. Biol. Chem. 258, 10794-10804(1983) (rat gamma-casein); Hall, Biochem. J. 242, 735-742 (1987) (humanalpha-lactalbumin); Stewart, Nucleic Acids Res. 12, 389 (1984) (bovinealpha S1 and kappa casein cDNAs); Gorodetsky et al., Gene 66, 87-96(1988) (bovine beta-casein); Alexander et al., Eur. J. Biochem. 178,395-401 (1988) (bovine kappa-casein); Brignon et al., FEBS Lett. 188,48-55 (1977) (bovine alpha S2 casein); Jamieson et al., Gene 61, 85-90(1987), Ivanov et al., Biol. Chem. Hoppe-Seyler 369, 425-429 (1988),Alexander et al., Nucleic Acids Res. 17, 6739 (1989) (bovine betalactoglobulin); Vilotte et al., Biochimie 69, 609-620 (1987) (bovinealpha-lactalbumin). The structure and function of the various milkprotein genes are reviewed by Mercier & Vilotte, J. Dairy Sci. 76,3079-3098 (1993). If additional flanking sequence are useful inoptimizing expression, such sequences can be cloned using the existingsequences, as probes. Mammary-gland specific regulatory sequences fromdifferent organisms can be obtained by screening libraries from suchorganisms using known cognate nucleotide sequences, or antibodies tocognate polypeptides, as probes.

According to this embodiment, the isolated nucleic acid may beoperatively associated with a milk-specific signal sequence, e.g., froma gene which encodes a product secreted into milk. For example, signalsequences from genes coding for caseins (e.g., alpha- beta-, gamma- orkappa-caseins), beta-lactoglobulin, whey acid protein, and lactalbuminare useful in the present invention.

The polypeptide can be expressed from an illustrative expressionconstruct that includes a promoter specific for mammary epithelialcells, e.g., a casein promoter (for example, a goat beta-caseinpromoter), a milk-specific signal sequence, e.g., a casein signalsequence (for example, beta-casein signal sequence), and a sequenceencoding the polypeptide.

The transgenic polypeptide can be produced in milk at relatively highconcentrations and in large volumes, providing continuous high leveloutput of normally processed polypeptide that is easily harvested from arenewable resource. There are several different methods known in the artfor isolation of polypeptides from milk.

Milk polypeptides usually are isolated by a combination of processes.Raw milk first is fractionated to remove fats, for example, by skimming,centrifugation, sedimentation (H. E. Swaisgood, Developments in DairyChemistry, I: Chemistry of Milk Protein, Applied Science Publishers, NY, 1982), acid precipitation (U.S. Pat. No. 4,644,056) or enzymaticcoagulation with rennin or chymotrypsin (Swaisgood, ibid.). Next, themajor milk proteins may be fractionated into either a clear solution ora bulk precipitate from which the specific protein of interest may bereadily purified.

U.S. Ser. No. 08/648,235 discloses a method for isolating a soluble milkcomponent, such as a protein, in its biologically active form from wholemilk or a milk fraction by tangential flow filtration. Unlike previousisolation methods, this eliminates the need for a first fractionation ofwhole milk to remove fat and casein micelles, thereby simplifying theprocess and avoiding losses of recovery and bioactivity. This method maybe used in combination with additional purification steps to furtherremove contaminants and purify the component of interest.

B. Production of Transgenic Polypeptides in the Eggs of a TransgenicAvian.

Recombinant polypeptide can also be produced in the eggs of a transgenicavian. e.g., a transgenic chicken, turkey, duck, goose, ostrich, guineafowl, peacock, partridge, pheasant, pigeon. Quail using methods known inthe art (Sang et al., Trends Biotechnology, 12:415-20, 1994). Genesencoding polypeptides specifically expressed in the egg, such asyolk-protein genes and albumin-protein genes, can be modified to directexpression of the lysosomal polypeptides of the invention.

Useful promoters for producing polypeptides in avian eggs are thosepromoters that are preferentially activated in the egg, includingpromoters that control the genes encoding egg polypeptides, e.g.,ovalbumin, lysozyme and avidin. Promoters from the chicken ovalbumin,lysozyme or avidin genes are preferred. Egg-specific promoters or thepromoters that are specifically activated in egg tissue can be from cDNAor genomic sequences.

DNA sequences of egg specific genes are known in the art (see, e.g.,Burley et al., “The Avian Egg”, John Wiley and Sons, p. 472, 1989, thecontents of which are incorporated herein by reference). Egg specificregulatory sequences from different organisms can be obtained byscreening libraries from such organisms using known cognate nucleotidesequences, or antibodies to cognate polypeptides, as probes.

C. Transgenic Plants.

Recombinant polypeptides can be expressed in a transgenic plant in whichthe transgene is inserted into the nuclear or plastidic genome. Planttransformation is known as the art. See, in general, Methods inEnzymology Vol. 153 (“Recombinant DNA Part D”) 1987, Wu and GrossmanEds., Academic Press and European Patent Application EP 693 554.

Foreign nucleic acids can be introduced into plant cells or protoplastsby several methods. For example, nucleic acid can be mechanicallytransferred by microinjection directly into plant cells by use ofmicropipettes Foreign nucleic acid can also be transferred into a plantcell by using polyethylene glycol which forms a precipitation complexwith the genetic material that is taken up by the cell (Paszkowski etal. (1984) EMBO J. 3:2712-22). Foreign nucleic acid can also beintroduced into a plant cell by electroporation (Fromm et al. (1985)Proc. Natl. Acad. Sci. USA 82:5824). In this technique, plantprotoplasts are electroporated in the presence of plasmids or nucleicacids containing the relevant genetic construct. Electrical impulses ofhigh field strength reversibly permeabilize biomembranes allowing theintroduction of the plasmids. Electroporated plant protoplasts reformthe cell wall, divide, and form a plant callus. Selection of thetransformed plant cells with the transformed gene can be accomplishedusing phenotypic markers.

Cauliflower mosaic virus (CaMV) can be used as a vector for introducingforeign nucleic acids into plant cells (Hohn et al. (1982) “MolecularBiology of Plant Tumors,” Academic Press, New York, pp. 549-560; Howell,U.S. Pat. No. 4,407,956). CaMV viral DNA genome is inserted into aparent bacterial plasmid creating a recombinant DNA molecule which canbe propagated in bacteria. The recombinant plasmid can be furthermodified by introduction of the desired DNA sequence. The modified viralportion of the recombinant plasmid is then excised from the parentbacterial plasmid, and used to inoculate the plant cells or plants.

High velocity ballistic penetration by small particles can be used tointroduce foreign nucleic acid into plant cells. Nucleic acid isdisposed within the matrix of small beads or particles, or on thesurface (Klein et al. (1987) Nature 327:70-73). Although typically onlya single introduction of a new nucleic acid segment is required, thismethod also provides for multiple introductions.

A nucleic acid can be introduced into a plant cell by infection of aplant cell, an explant, a meristem or a seed with Agrobacteriumtumefaciens or Agrobacterium rhizogenes transformed with the nucleicacid. Under appropriate conditions, the transformed plant cells aregrown to form shoots, roots, and develop further into plants. Thenucleic acids can be introduced into plant cells, for example, by meansof the Ti plasmid of Agrobacteria. The Ti plasmid is transmitted toplant cells upon infection by Agrobacteria, and is stably integratedinto the plant genome (Horsch et al. (1984) “Inheritance of FunctionalForeign Genes in Plants,” Science 233:496498; Fraley et al. (1983) Proc.Natl. Acad. Sci. USA 80:4803).

Plants from which protoplasts can be isolated and cultured to give wholeregenerated plants can be transformed so that whole plants are recoveredwhich contain the transferred foreign gene. Some suitable plantsinclude, for example, species from the genera Fragaria, Lotus, Medicago,Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium,Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa,Capsicum, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia.Digitalis, Majorana, Ciohorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Hererocallis, Nemesia, Petargonium, Panicum, Pennisetum,Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Lolium,Zea, Triticum, Sorghum, and Datura.

Plant regeneration from cultured protoplasts is described in Evans etal., “Protoplasts Isolation and Culture,” Handbook of Plant CellCultures 1:124-176 (MacMillan Publishing Co. New York 1983); M. R.Davey, “Recent Developments in the Culture and Regeneration of PlantProtoplasts,” Protoplasts (1983)-Lecture Proceedings, pp. 12-29.(Birkhauser, Basal 1983); P. J. Dale, “Protoplast Culture and PlantRegeneration of Cereals and Other Recalcitrant Crops,” Protoplasts(1983)-Lecture Proceedings, pp. 31-41, (Birkhauser, Basel 1983); and H.Binding. “Regeneration of Plants,” Plant Protoplasts, pp. 21-73, (CRCPress. Boca Raton 1985).

Regeneration from protoplasts varies from species to species of plants,but generally a suspension of transformed protoplasts containing copiesof the exogenous sequence is first generated. In certain species, embryoformation can then be induced from the protopiast suspension, to thestage of ripening and germination as natural embryos. The culture mediacan contain vanous amino acids and hormones, such as auxin andcytokinins. It can also be advantageous to add glutamic acid and prolineto the medium, especially for such species as corn and alfalfa. Shootsand roots normally develop simultaneously. Efficient regeneration willdepend on the medium, on the genotype, and on the history of theculture. If these three variables are controlled, then regeneration isfully reproducible and repeatable.

In vegetatively propagated crops, the mature transgenic plants can bepropagated by the taking of cuttings or by tissue culture techniques toproduce multiple identical plants for trialling, such as testing forproduction characteristics. Selection of a desirable transgenic plant ismade and new varieties are obtained thereby, and propagated vegetativelyfor commercial sale. In seed propagated crops, the mature transgenicplants can be self crossed to produce a homozygous inbred plant. Theinbred plant produces seed containing the transgene. These seed can begrown to produce plants that have the selected phenotype. The inbredsaccording to this invention can be used to develop new hybrids. In thismethod a selected inbred line is crossed with another inbred line toproduce the hybrid.

Parts obtained from a transgenic plant, such as flowers, seeds, leaves,branches, fruit, and the like are covered by the invention, providedthat these parts include cells which have been so transformed. Progenyand variants, and mutants of the regenerated plants are also includedwithin the scope of this invention, provided that these parts comprisethe introduced nucleic acid sequences. Progeny and variants, and mutantsof the regenerated plants are also included within the scope of thisinvention.

Selection of transgenic plants or plant cells can be based upon a visualassay, such as observing color changes (e.g., a white flower, variablepigment production, and uniform color pattern on flowers or irregularpatterns), but can also involve biochemical assays of either enzymeactivity or product quantitation. Transgenic plants or plant cells aregrown into plants bearing the plant part of interest and the geneactivities are monitored, such as by biochemical assays (Northernblots); Western blots; and enzyme assays. Appropriate plants areselected and further evaluated. Methods for generation of geneticallyengineered plants are further described in U.S. Pat. Nos. 5,283,184,5,482,852, and European Patent Application EP 693 554, all of which areincorporated herein by reference.

V. Subjects. Pharmaceutical Formulations. Vaccine and Modes ofAdministration

The present invention finds use in veterinary and medical applications.Suitable subjects include both avians and mammals, with mammals beingpreferred. The term “avian” as used herein includes, but is not limitedto, chickens, ducks, geese, quail, turkeys and pheasants. The term“mammal” as used herein includes, but is not limited to, humans,non-human primates, bovines, ovines, caprines, equines, felines,canines, lagomorphs, rats, mice etc. Human subjects are preferred. Humansubjects include neonates, infants, juveniles, and adults. Inrepresentative embodiments, the subject is a human subject that has oris believed to have a lysosomal polypeptide (e.g., GAA) deficiency.

In particular embodiments, the present invention provides apharmaceutical composition comprising an isolated nucleic acid or vectorof the invention in a pharmaceutically-acceptable carrier and,optionally, other medicinal agents, pharmaceutical agents, carriers,adjuvants, dispersing agents, diluents, and the like. For injection, thecarrier will typically be a liquid, such as sterile pyrogen-free water,pyrogen-free phosphate-buffered saline solution, bacteriostatic water,or Cremophor EL[R] (BASF, Parsippany, N.J.). For other methods ofadministration, the carrier may be either solid or liquid.

By “pharmaceutically acceptable” it is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to a subject along with the isolated nucleic acid or vectorwithout causing any undesirable biological effects such as toxicity.Thus, such a pharmaceutical composition can be used, for example, intransfection of a cell ex vivo or in administering an isolated nucleicacid or vector directly to a subject.

In the case of a viral vector, virus particles may be contacted with thecells at the appropriate multiplicity of infection according to standardtransduction methods appropriate for the particular target cells Titersof virus to administer can vary, depending upon the target cell type andthe particular virus vector, and can be determined by those of skill inthe art without undue experimentation. Typically, at least about 10³virus particles, at least about 10⁵ particles, at least about 10⁷particles, at least about 10⁹ particles, at least about 10¹¹ particles,or at least about 10¹² particles are administered to the cell. Inexemplary embodiments, about 10⁷ to about 10¹⁵ particles, about 10⁷ toabout 10¹³ particles, about 10⁸ to about 10¹² particles, about 10¹⁰ toabout 10¹⁵ particles, about 10¹¹ to about 10¹⁵ particles, about 10¹² toabout 10¹⁴ particles, or about 10¹² to about 10¹³ particles areadministered.

The cell to be administered the vectors of the invention can be of anytype, including but not limited to neuronal cells (including cells ofthe peripheral and central nervous systems), retinal cells, epithelialcells (including dermal, gut, respiratory, bladder, pulmonary,peritoneal and breast tissue epithelium), muscle (including cardiac,smooth muscle, including pulmonary smooth muscle cells, skeletal muscle,and diaphragm muscle), pancreatic cells (including islet cells), hepaticcells (including parenchyma), cells of the intestine, fibroblasts (e.g.,skin fibroblasts such as human skin fibroblasts), fibroblast-derivedcells, endothelial cells, intestinal cells, germ cells, lung cells(including bronchial cells and alveolar cells), prostate cells, stemcells, progenitor cells, dendritic cells, and the like. Alternatively,the cell is a cancer cell (including tumor cells). Moreover, the cellscan be from any species of origin, as indicated above.

Mammalian cells include but are not limited to CHO cells, 293 cells,HT1080 cells, HeLa cells or C10 cells.

In particular embodiments of the invention, the cell has been removedfrom a subject, the vector is introduced therein, and the cell is thenreplaced back into the subject. Methods of removing cells from subjectsfor treatment ex vivo, followed by introduction back into the subjectare known in the art (see, e.g., U.S. Pat. No. 5,399,346 for theteaching of ex vivo virus vector administration). As a furtheralternative, the cells that are manipulated and then introduced into thesubject are provided from another subject or cell line as a cell-basedform of therapy.

A further aspect of the invention is a method of treating subjects invivo with the inventive nucleic acids or delivery vectors.Administration of the nucleic acid or delivery vectors of the presentinvention to a human subject or an animal can be by any means known inthe art. The subject can be a mammalian subject, more particularly ahuman subject. In other embodiments, the subject is in need oftreatment, for example, has been diagnosed with or is suspected ofhaving a lysosomal polypeptide (e.g., GAA) deficiency.

Dosages will depend upon the mode of administration, the severity of thedisease or condition to be treated, the individual subject's condition,the particular vector, and the gene to be delivered, and can bedetermined in a routine manner (see, e.g., Remington, The Science AndPractice of Pharmacy (9^(th) Ed. 1995)). In particular embodiments, theisolated nucleic acid or vector is administered to the subject in atherapeutically effective amount, as that term is defined above.

Typically, with respect to viral vectors, at least about 10³, at leastabout 10⁵, at least about 10⁷, at least about 10⁹, at least about 10¹¹virus particles, or at least about 10¹² virus particles are administeredto the subject per treatment. Exemplary doses are virus titers of about10⁷ to about 10¹⁵ particles, about 10⁷ to about 10¹⁴ particles, about10⁸ to about 10¹³ particles, about 10¹⁰ to about 10¹⁵ particles, about10¹¹ to about 10¹⁵ particles, about 10¹² to about 10¹⁴ particles, orabout 10¹² to about 10¹³ particles.

In particular embodiments of the invention, more than one administration(e.g., two, three, four, or more administrations) may be employed toachieve therapeutic levels of nucleic acid expression.

Exemplary modes of administration include oral, rectal, transmucosal,topical, transdermal, inhalation, parenteral, e.g., intravenous,subcutaneous, intradermal, intramuscular (i.e., administration tocardiac, skeletal, diaphragm and/or smooth muscle), and intraarticularadministration, and the like, as well as direct tissue (e.g., muscle) ororgan injection (e.g., into the liver, into the brain for delivery tothe central nervous system), alternatively, intrathecal, directintramuscular (e.g. into cardiac, skeletal or diaphragm muscle),intraventricular intravenous, intraperitoneal, intranasal, orintraocular injections. Administration to the liver (discussed below) isanother representative mode of administration.

Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. An injectionmedium will typically be an aqueous liquid that contains the additivesusual for injection solutions, such as stabilizing agents, salts orsaline, and/or buffers.

For oral administration, the isolated nucleic acid or vector can beadministered in solid dosage forms, such as capsules, tablets, andpowders, or in liquid dosage forms, such as elixirs, syrups, andsuspensions. Active component(s) can be encapsulated in gelatin capsulestogether with inactive ingredients and powdered carriers, such asglucose, lactose, sucrose, mannitol, starch, cellulose or cellulosederivatives, magnesium stearate, stearic acid, sodium saccharin, talcum,magnesium carbonate and the like. Examples of additional inactiveingredients that may be added to provide desirable color, taste,stability, buffering capacity, dispersion or other known desirablefeatures are red iron oxide, silica gel, sodium lauryl sulfate, titaniumdioxide, edible white ink and the like. Similar diluents can be used tomake compressed tablets. Both tablets and capsules can be manufacturedas sustained release products to provide for continuous release ofmedication over a period of hours. Compressed tablets can be sugarcoated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric-coated for selectivedisintegration in the gastrointestinal tract. Liquid dosage forms fororal administration can contain coloring and flavoring to increasepatient acceptance.

Oral administration of AAV vectors has been described by U.S. Pat. No.6,110,456 (incorporated by reference herein in its entirety).

The isolated nucleic acid or vector may alternatively be formulated fornasal administration or otherwise administered to the lungs of a subjectby any suitable means, but is preferably administered by an aerosolsuspension of respirable particles comprising the vector which thesubject inhales. The respirable particles may be liquid or solid. Theterm “aerosol” includes any gas-borne suspended phase, which is capableof being inhaled into the bronchioles or nasal passages. Specifically,aerosol includes a gas-borne suspension of droplets, as may be producedin a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosolalso includes a dry powder composition suspended in air or other carriergas, which may be delivered by insufflation from an inhaler device, forexample. See Ganderton & Jones, Drug Delivery to the Respiratory Tract,Ellis Horwood (1987); Gonda (1990) Critical Reviews in Therapeutic DrugCarrier Systems 6:273-313; and Raeburn et al. (1992) J. Pharmacol.Toxicol. Methods 27:143-159. Aerosols of liquid particles comprising theisolated nucleic acid or vector may be produced by any suitable means,such as with a pressure-driven aerosol nebulizer or an ultrasonicnebulizer, as is known to those of skill in the art. See, e.g., U.S.Pat. No. 4,501,729. Aerosols of solid particles comprising the isolatednucleic acid or vector may likewise be produced with any solidparticulate medicament aerosol generator, by techniques known in thepharmaceutical art.

Alternatively, one may administer the isolated nucleic acid or vector ina local rather than systemic manner, for example, in a depot orsustained-release formulation.

In particular embodiments of the invention, the isolated nucleic acid orvector is delivered to the liver of the subject. Administration to theliver can be achieved by any method known in the art, including, but notlimited to intravenous administration, intraportal administration,intrabiliary administration, intra-arterial administration, injectioninto the liver parenchyma, and intrasplenic injection.

Intramuscular delivery and intracardiac delivery to skeletal muscle orcardiac muscle, respectively, or direct injection into diaphragm muscleis also preferred. In other particular embodiments, intraperitonealadministration is used to deliver the isolated nucleic acid or vector todiaphragm muscle.

In particular embodiments, the isolated nucleic acid (e.g., carried byan Ad, AAV or hybrid Ad/AAV vector) encoding a lysosomal polypeptide isintroduced into a depot organ or tissue (e.g., liver, skeletal muscle,lung) and the polypeptide is expressed therein and secreted into thecirculatory system, where it is optionally delivered to target tissues,preferably, in a therapeutically effective amount. Intramusculardelivery to skeletal muscle or delivery to the liver are illustrativefor the practice of this embodiment of the invention. Alternatively, theisolated nucleic acid or vector can be administered to the brain (e.g.,to treat MPS disorders such as Sly disease), where the polypeptide canbe expressed and secreted by transformed or transduced cells (e.g.,neurons, glial cells) and taken up by other brain cells.

Having now described the invention, the same will be illustrated withreference to certain examples, which are included herein forillustration purposes only, and which are not intended to be limiting ofthe invention.

Example 1 Materials and Methods

Cell Culture.

293 cells and C-7 cells (Amalfitano and Chamberlain. (1997) Gene Ther.4:258-263) were maintained in Dulbecco's modified Eagle mediumsupplemented with 10% fetal bovine serum, 100 U penicillin permilliliter, and 100 μg streptomycin per milliliter at 37° C. in a 5%CO-air atmosphere. C-7 cells were grown in the presence of hygromycin,50 μg/ml. HeLa cells were maintained in minimum essential medium Eaglesupplemented with 10% fetal bovine serum, 1 mM minimum essential mediumsodium pyruvate, 0.1 mM minimum essential medium nonessential aminoacids, 100 U penicillin per milliliter, and 100 μg streptomycin permilliliter at 37° C. in a 5% CO₂-air atmosphere.

Construction of an AAV Vector Plasmid Encoding hGAA.

The hGAA cDNA was subcloned with the CMV promoter from pcDNA3-hGAA (VanHove et al. (1996). Proc. Natl. Acad. Sci. USA 93:65-70) into an AAVvector plasmid, as an NruI-EcoRV fragment, upstream of the human growthhormone intron 4 and polyadenylation sequence (Brinster et al. (19881Proc. Natl Acad. Sci. USA 85: 836-840). The resulting transcriptionalunit was flanked by the AAV2 terminal repeat (TR) sequences inpAAV-ChGAAGH. A 530 bp deletion spanning the human growth hormone intron4 was generated by EcoRV and partial PvuII digestion followed byblunting of ends with T4 DNA polymerase and ligation with T4 DNA ligaseto generate pAAV-ChGAAG(−). The hybrid CMV enhancer/chicken β-actin (CB)promoter was amplified by polymerase chain reaction from pTriEx1(Novagen, Madison, Wis.) with primers that introduced unique upstreamXbaI and downstream KpnI restriction sites, and the CB promoter wassubcloned as a KpnI-XbaI fragment to replace the CMV promoter inpAAV-ChGAAG(−), to generate pAAV-CBhGAAG(−). Next, in order to reducethe packaging size further, the plasmid pAAV-CBhGAAG(−) was linearizedat a unique AflII site in the 3′ untranslated sequence of the hGAA cDNAand partially digested with NspI to introduce a 411 bp deletion in the3′ untranslated sequence of the hGAA cDNA, followed by blunting of endswith T4 DNA polymerase and ligation with T4 DNA ligase to generatepAAV-CBGAApA. Finally, the vector sequences from pAAV-CBGAApA wereisolated as a 4.4 kbp fragment from a partial BglII digest, and ligatedwith the calf intestinal alkaline phosphatase-dephosphorylated BglIIsite of pShuttle (He et al. (1998). Proc. Natl. Acad. Sci. USA95:2509-2514).

Construction of a Hybrid [E1-, Polymerase-, Preterminal Protein-]Ad-AAVVector Encoding hGAA.

Kanamycin-resistant shuttle plasmids were constructed to contain withinthe Ad E1 region the CB promoter+hGAA cDNA+polyA transgene cassetteflanked by the AAV2 TR sequences. The shuttle plasmid was digested withPmeI, and electroporated into the BJ5183 recombinogenic strain of E.Coli with the pAd[E1−, polymerase−, preterminal protein−] plasmid(Hodges et al. (2000). J. Gene Med. 2:250-259). Recombinantkanamycin-resistant clones were screened by restriction enzyme digestion(BstXI) to confirm successful generation of the full-length recombinantAd vector genomes. These clones were digested with Pad and transfectedas previously described into the E1 and E2b expressing cell line, C-7(Hodges et al. (2000). J. Gene Med. 2:250-259). The vectors wereamplified and confirmed to have the correct construction by restrictionenzyme mapping of vector genomes, and subsequent functional assays invitro and in vivo. Once isolated, the respective Ad vectors are seriallypropagated in increasing numbers of C-7 cells (Amalfitano et al. (1998)J. Virol. 72:926-933). Forty-eight hours after infection, infected cellpellets were harvested by low speed centrifugation, resuspended in 10 mMTris-HCl pH 8.0, vector released from the cells by repeatedfreeze-thawing (×3) of the lysate, released by ultrasonification, andthe vector containing supernatant subjected to two rounds of equilibriumdensity CsCl centrifugation (Amalfitano et al. (1998) J. Virol.72:926-933). Two virus bands were visible. The virus bands were thenremoved, dialyzed extensively against 10 mM Tris-HCl pH 8.0 (or PBS),sucrose added to 1%, and aliquots stored at −80° C. The number of vectorparticles was quantified based on the OD₂₆₀ of vector contained indialysis buffer with sodium dodecyl sulfate (SDS) disruption, and byDNase I digestion, DNA extraction, and Southern blot analysis.

Hybrid Ad-AAV vector DNA analysis consisted of vector DNA isolation andrestriction enzyme digestion followed by Southern blotting to verify thepresence of intact AAV vector sequences within the lower band in thecesium chloride gradient, including restriction enzymes thatdemonstrated the presence of AAV terminal repeat sequences flanking thetransgene (AhdI and BssHII).

All viral vector stocks were handled according to Biohazard Safety Level2 guidelines published by the NIH.

In Vivo Administration of Hybrid Ad-AAV or AAV Vector Stocks.

The vector was administered intramuscularly into both gastrocnemiusmuscles of 3-day-old GAA-KO mice (Raben et al. (1998) J. Biol. Chem.273:19086-19092). A total of 4×10¹⁰ DNase 1-resistant Ad-AAV vectorparticles were administered, divided between 2 equal injections peranimal. Alternatively, a total of 10¹¹ DNase I-resistant AAV vectorparticles was administered intramuscularly in the right gastrocnemiusmuscle of 6 week-old GAA-KO/SCID mice. For liver-targetedadministration, a total of 10¹¹ DNase I-resistant AAV vector particleswas administered intravenously via the retroorbital sinus or via theportal vein to 3 month-old GAA-KO/SCID or GAA-KO mice as indicated. Atthe respective time points post-injection, plasma or tissue samples wereobtained and processed as described below. All animal procedures weredone in accordance with Duke University institutional Animal Care andUse Committee-approved guidelines.

Determination of hGAA Activity and Glycogen Content.

Tissue hGAA activity was measured following removal of the tissue fromcontrol or treated mice, flash-freezing on dry ice, homogenization andsonication in distilled water, and pelleting of insolublemembranes/proteins by centrifugation. The protein concentrations of theclarified suspensions were quantified via the Bradford assay, hGAAactivity in the muscle was determined as described (Amalfitano et al.(1999) Proc. Natl. Acad. Sci. USA 96:8861-8866). Glycogen content oftissues was measured using the Aspergillus niger assay system, asdescribed (Kikuchi et al. (1998) J. Clin. Invest. 101:827-833). Atwo-tailed homscedastic Student's t-test was used to determinesignificant differences in hGAA levels and glycogen content betweenGAA-KO mice with or without administration of the vector encoding hGAA.

Western Blotting Analysis of hGAA.

For direct detection of hGAA in tissues, samples (100 μg of protein)were electrophoresed overnight in a 6% polyacrylamide gel to separateproteins, and transferred to a nylon membrane. The blots were blockedwith 5% nonfat milk solution, incubated with primary and secondaryantibodies and visualized via the enhanced chemiluminescence (ECL)detection system (Amersham Pharmacia, Piscataway, N.J.).

ELISA Detection of Plasma Anti-hGAA and Anti-Ad Antibodies.

Recombinant hGAA (5 μg) in carbonate buffer was coated onto each well ofa 96-well plate at 4° C. overnight. Alternatively, the wells were coatedwith 5×10⁸ Ad vector particles per well at 4° C. overnight for detectionof anti-Ad antibody. After washing with phosphate buffered saline (PBS)containing 0.05% Tween 20, serial dilutions of the plasma were added tothe wells, and incubated for 1 hour at room temperature. The wells werewashed with 0.05% Tween 20+PBS, incubated with a 1:2,500 dilution ofalkaline phosphatase-conjugated sheep anti-mouse IgG (H+L) at roomtemperature for 1 hour, washed, and alkaline phosphatase substrate(p-nitrophenyl phosphate) added. The absorbance values of the plateswere read at 405 nm with a Bio-Rad microplate reader ELISA (all samplesyielded absorbance values that were within the linear range of the assayat this dilution) (Ding et al. (2002) Mol. Ther. 5:436-446). The titerof antibody was determined as the highest dilution where the value forabsorbance exceeded 0.1.

Example 2 Neonatal Muscle-Targeted Ad-AAV Administration and hGAAProduction in GAA-KO Mice

Muscle-targeted expression of hGAA with an Ad-AAV hybrid vector wasevaluated in neonatal GAA-KO mice. While the Ad-AAV vector deliveredmuscle-targeted hGAA in these experiments, the hybrid Ad-AAV vector inquestion was developed to provide improved AAV vector packagingefficiency. Administration of the Ad-AAV vector in 3 day-old micereversed the effects of GSD II within the injected gastrocnemius muscle,and sustained hGAA expression provided long-term therapeutic resultsevidenced by generally reduced glycogen storage in the muscles of thehind limb.

The Ad-AAV vector encoding hGAA was targeted to both gastrocnemiusmuscles by intramuscular injection on day of life 3 in GAA-KO mice, andhGAA levels were analyzed at 6, 12, and 24 weeks of age. Western blotanalysis of hGAA in the gastrocnemius, hamstrings and quadriceps musclegroups at 24 weeks of age showed hGAA of the ˜110 kD, 76 kD and 67 kDisoforms following Ad-AAV vector administration, and hGAA was absent forthe skeletal muscle of untreated GAA-KO mice (FIG. 1 Panel A).Apparently due to transduction of muscle adjacent to the gastrocnemius,the highest amount of hGAA was seen in the hamstrings, with slightlylower hGAA in the gastrocnemius and the lowest hGAA in the quadriceps. Asimilar pattern of introduced hGAA was detected by Western blot analysisof gastrocnemius and quadriceps muscle groups following Ad-AAV vectoradministration at 6 and 12 weeks of age (not shown).

Western blot analysis of hGAA in heart, diaphragm, and liver at 6, 12,and 24 weeks of age following neonatal Ad-AAV vector administrationdemonstrated low, detectable levels of hGAA for the ˜76 kD isoform ofhGAA in 2 of 4 GAA-KO mice at 6 weeks (FIG. 1 Panel B; m1 and m4). LowerhGAA was present in the heart for 1 of 4 GAA-KO mice at 24 weeks (FIG. 1Panel B; m12), and was not detected in the heart for 5 GAA-KO mice at 12weeks of age (FIG. 1 Panel B) Finally, hGAA was not detected in plasmaby Western blot at 3 weeks following vector administration (not shown).

The function of hGAA introduced by the Ad-AAV vector was analyzed by GAAenzyme assay, and GAA activity correlated with the relative amounts ofhGAA protein detected by Western blot analysis (FIG. 2 Panel A). At 24weeks, the highest GAA activity was present in hamstrings (1180+/−620nmol/mg/hr), followed by gastrocnemius (717+/−275 nmol/mg/hr), andlowest in quadriceps (44+/−28 nmol/mg/hr). GAA activity was elevated inall muscle groups at 6, 12, and 24 weeks, compared to the same musclegroups in untreated, GAA-KO mice (FIG. 2 Panel B). The hGAA activity forhamstrings was approximately 50-fold elevated compared to the GAAactivity in wild-type mouse skeletal muscle (Ding, E., et al. (2002)Mol. Ther. 5:436-446).

GAA activity was elevated in heart, diaphragm and liver at 6 weeks ofage following Ad-AAV vector administration in 2 of 4 GAA-KO mice (FIG. 2Panel B; ml and m4). However, GAA activity in these tissues was muchlower than for skeletal muscles in the hindleg closer to the site ofAd-AAV vector injection (FIG. 2 Panel B).

Example 3 Anti-hGAA Antibodies Following Neonatal Ad-AAV Administration

Anti-hGAA antibody formation occurred during hGAA enzyme replacement inGSD II during a clinical trial (Amalfitano et al. (2001) Genet. In Med.3:132-138) and in preclinical use of adenoviral vectors in GAA-KO mice(Ding et al. (2002) Mol. Ther. 5:436-446). Anti-hGAA antibodies weredetected in GAA-KO mice at 6, 12, and 24 weeks after neonatal Ad-AAVvector administration (FIG. 3 Panel A, Neonatal Intramuscular Ad-AAV),with 3 exceptions, while anti-hGAA antibodies were absent in untreatedGAA-KO mice (FIG. 3 Panel A, Control). Adult GAA-KO mice that receivedthe Ad-AAV vector intravenously were used as positive controls (FIG. 3Panel A, Ad-AAV). These mice developed high-titer antihGAA antibodies asreported previously (Ding. E., et al. (2002) Mol. Ther. 5:436-446). Thepresence of hGAA antibodies at a titer of >1:4,000 was demonstrated for10 of 13 mice following neonatal Ad-AAV vector administration (FIG. 3Panel B, Neonatal Intramuscular Ad-AAV), and for 3 of 3 adult GAA-KOmice following intravenous Ad-AAV vector administration (FIG. 3 Panel B,Ad-AAV). Only the 3 GAA-KO mice that failed to generate anti-hGAAantibodies following neonatal vector administration (ml, m4, and m12 inFIG. 3 Panel B) also featured significant hGAA by Western blot analysisin heart at 6 and 24 weeks of age (FIG. 1 Panel B). The formation ofanti-hGAA antibodies appeared to be related to the persistent expressionof the foreign transgene (hGAA), because GAA-KO mice failed to generateanti-Ad antibodies following neonatal, one-time exposure to the Ad-AAVvector (FIG. 3 Panel C).

Example 4 Reduced Glycogen Content in Skeletal Muscles of the Hind LimbFollowing Injection of the Gastrocnemius in Neonatal GAA-KO Mice

The benefit of introduced hGAA in GSD II was shown by glycogenquantitation in skeletal muscle. Glycogen storage was reducedsignificantly for skeletal muscle groups at all time points except forthe quadriceps at 24 weeks (FIG. 4 Panel A), when hGAA in the quadricepswas not as high as for other timepoints (FIG. 2 Panel A). Glycogencontent was significantly reduced for the hamstrings, gastrocnemius, andquadriceps compared to untreated GAA-KO mice (FIG. 4 Panel A). Glycogencontent was reduced to 0.77+/−0.42 mmol glucose/gm protein in the heartof GAA-KO mice (n=4) 6 weeks after neonatal Ad-AAV vectoradministration, compared to 2.1+/1.3 mmol glucose/gm protein inuntreated GAA-KO mice (n=3). Only 2 of 4 GAA-KO mice had detectableheart and diaphragm hGAA at 6 weeks by Western blot analysis (FIG. 1Panel B; ml and m4), and both the heart and diaphragm glycogen contentwas somewhat reduced in 1 of those mice (FIG. 4 Panel B; m4).

The correction of glycogen storage by introduced hGAA was evident byglycogen staining of gastrocnemius and heart. Periodic-acid Schiff (PAS)staining of gastrocnemius showed much less lysosomal accumulation ofglycogen at 24 weeks following Ad-AAV vector administration compared toan untreated GAA-KO mouse (FIG. 5). In a GAA-KO mouse that hadsignificant hGAA in heart at 6 weeks (FIG. 1 Panel B; m4), the glycogenaccumulation in heart was less than for an untreated, GAA-KO mouse (FIG.5). In aggregate, these data provide evidence for the continued benefitof hGAA introduced with a vector in muscle.

Example 5 Construction of an AAV Vector Plasmid Encoding hGAA

The hGAA cDNA was subcloned with the CMV promoter from pcDNA3-hGAA (VanHove et al. (1996). Proc. Natl. Acad. Sci. USA 93:65-70) into an AAVvector plasmid, as an NruI-EcoRV fragment, upstream of the human growthhormone intron 4 and polyadenylation sequence (Brinster et al. (1988).Proc. Natl. Acad. Sci. USA 85: 836-840). The resulting transcriptionalunit was flanked by the AAV2 TR sequences in pAAV-ChGAAGH. A 530 bpdeletion spanning the human growth hormone intron 4 was generated byEcoRV and partial PvuII digestion followed by blunting of ends withT4′DNA polymerase and ligation with T4 DNA ligase to generatepAAV-ChGAAG(−). The hybrid CMV enhancer/chicken β-actin (CB) promoterwas amplified by polymerase chain reaction from pTriEx1 (Novagen,Madison, Wis.) with primers that introduced unique upstream XbaI anddownstream KpnI restriction sites, and the CE promoter was subcloned asa KpnI-XbaI fragment to replace the CMV promoter in pAAV-ChGAAG(−), togenerate pAAV-CBhGAAG(−). in order to reduce the packaging size further,the plasmid pAAV-CBhGAAG(−) was linearized at a unique AflII site in the3′ untranslated sequence of the hGAA cDNA and partially digested withNspI to introduce a 411 bp deletion in the 3′ untranslated sequence ofthe hGAA cDNA, followed by blunting of ends with T4 DNA polymerase andligation with T4 DNA ligase to generate pAAV-CBGAApA. Finally, thevector sequences from pAAV-CBGAApA were isolated as a 4.4 kbp fragmentfrom a partial BglII digest, and ligated with the calf intestinalalkaline phosphatase-dephosphorylated BglII site of pShuttle (He, T.-C.,et al. (1998). Proc. Natl. Acad. Sci. USA 95:2509-2514).

Example 6 Construction of a Hybrid [E1−, Polymerase-, PreterminalProtein-] Ad-AAV Vector Encoding hGAA

Kanamycin-resistant shuttle plasmids were constructed to contain withinthe Ad E1 region the CB promoter+hGAA cDNA-polyA transgene cassetteflanked by the AAV2 TR sequences. The shuttle plasmid was digested withPmeI, and electroporated into the BJ5183 recombinogenic strain of E.Coli with the pAd[E1−, polymerase−, preterminal protein−] plasmid(Hodges et al. (2000). J. Gene Med. 2:250-259). Recombinantkanamycin-resistant clones were screened by restriction enzyme digestion(BstXI) to confirm successful generation of the full-length recombinantAd vector genomes. These clones were digested with PacI and transfectedas previously described into the E1, and E2b expressing cell line, C-7(Amalfitano et al. (1998). J. Virol. 72:926-933).

The vectors were amplified and confirmed to have the correctconstruction by restriction enzyme mapping of vector genomes, andsubsequent functional assays in vitro and in vivo. Once isolated, therespective Ad vectors are serially propagated in increasing numbers ofC-7 cells (Amalfitano et al. (1998). J. Virol. 72:926-933). Forty-eighthours after infection, infected cell pellets were harvested by low speedcentrifugation, resuspended in 10 mM Tris-HCl pH 8.0. vector releasedfrom the cells by repeated freeze-thawing (×3) of the lysate, releasedby ultrasonification, and the supernatant containing vector wassubjected to two rounds of equilibrium density CsCl centrifugation(Amalfitano et al. (1998). J. Virol. 72:926-933). Two virus bands werevisible. The virus bands were then removed, dialyzed extensively against10 mm Tris-HCl pH 8.0 (or PBS), sucrose added to 1%, and aliquots storedat −80° C. The number of vector particles was quantified biased on theOD₂₆₀ of vector contained in dialysis buffer with sodium dodecyl sulfate[SDS] disruption, and by DNase I digestion, DNA extraction, and Southernblot analysis.

Hybrid Ad-AAV vector DNA analysis consisted of vector DNA isolation andrestriction enzyme digestion followed by Southern blotting to verify thepresence of intact AAV vector sequences within the lower band in thecesium chloride gradient, including restriction enzymes thatdemonstrated the presence of AAV terminal repeat sequences flanking thetransgene (AhdI and BssHII) (Sun et al., (2003) Mol Ther 7:193-201).

Example 7 Preparation of AAV Vectors

AAV vector stocks were prepared as described herein with modificationsas described (Sun B D, Chen Y-T, Bird A, Xu F, Hou Y-X, Amalfitano A,and Koeberl D D. Packaging of an AAV vector encoding human acidα-glucosidase for gene therapy in glycogen storage disease type II witha modified hybrid adenovirus-adeno-associated virus vector. Mol Ther7:467-477, 2003; Halbert C L, Allen J M, Miller A D. Adeno-associatedvirus type 6 (AAV) vectors mediate efficient transduction of airwayepithelial cells in mouse lungs compared to that of AAV2 vectors.(Halbert et al., (2001) J Virol 75:6615-6624). Briefly, 293 cells wereinfected with the hybrid Ad-AAV vector (2000 DNase I-resistant vectorparticles/cell as quantitated by Southern blot analysis) containing theAAV vector sequences 15-30 minutes before transfection with an AAVpackaging plasmid containing the AAV2 Rep and AAV2 or AAV6 (for AAV6vector [Halbert et al. (2001) J. Virol. 75:6615-6624]). Cap genes weredriven by heterologous promoters, which typically generate no detectablereplication-competent AAV (rcAAV) (Allen et al. (2000) Mol. Ther.1:88-95; Allen et al. (1997) J. Virol. 71:6816-6822). Cell lysate washarvested 48 hours following infection and freeze-thawed 3 times,isolated by iodixanol step gradient centrifugation before heparinaffinity column purification (Zolotukhin et al. (1999) Gene Ther.6:973-995; Halbert C L, Allen J M, Miller A D. Adeno-associated virustype 6 (AAV) vectors mediate efficient transduction of airway epithelialcells in mouse lungs compared to that of AAV2 vectors. J Virol 2001;75:6615-6624), and aliquots were stored at −80° C. The number of vectorDNA containing-particles was determined by DNase I digestion, DNAextraction, and Southern blot analysis. Contaminating wt AAV particleswere detected in recombinant AAV vector preparations by Southern blotanalysis of extracted vector DNA, and by a sensitive PCR assay utilizingprimers spanning the junction between the rep and cap genes. The levelof rcAAV was less than 1 particle in 10⁵ AAV vector particles. All viralvector stocks were handled according to Biohazard Safety Level 2guidelines published by the NIH.

Example 8 In Vivo Administration of AAV Vector Stocks

AAV vector was administered intramuscularly into the gastrocnemiusmuscle of 6 week-old GAA-KO mice (Raben et al. (1998) J. Biol. Chem.273:19086-19092)/SCID mice. One hundred μl containing 1×10¹¹ DNaseI-resistant AAV vector particles were injected per gastrocnemius. Forportal vein injection, an AAV vector was administered via portal veininjection. At the respective time points post-injection, plasma ortissue samples were obtained and processed as described below. Forintravenous administration, an AAV vector was administered via theretroorbital sinus. All animal procedures were done in accordance withDuke University Institutional Animal Care and Use Committee-approvedguidelines.

Example 9 Determination of hGAA Activity and Glycogen Content

Tissue hGAA activity was measured following removal of the tissue fromcontrol or treated mice, flash-freezing on dry ice, homogenization andsonication in distilled water, and pelleting of insolublemembranes/proteins by centrifugation. Untreated, affected controls wereGAA-KO mice, 12 weeks old at the time of analysis except where notedotherwise. The protein concentrations of the clarified suspensions werequantified via the Bradford assay. hGAA activity in the muscle wasdetermined as described (Kikuchi, T., et al. (1998) J. Clin. Invest.101:827-833). Glycogen content of tissues was measured using theAspergillus niger assay system, as described (Amalfitano et al. (1999)Proc. Natl. Acad. Sci. USA 96:8861-8866). A two-tailed homoscedasticStudent's t-test was used to determine significant differences in hGAAlevels and glycogen content between GAA-KO, mice with or withoutadministration of the vector encoding hGAA.

Example 10 Western Blotting Analysis of hGAA

For direct detection of hGAA in tissues, samples (100 μg of protein)were electrophoresed overnight in a 6% polyacrylamide gel to separateproteins, and transferred to a nylon membrane. The blots were blockedwith 5% nonfat milk solution, incubated with primary and secondaryantibodies and visualized via the enhanced chemiluminescence (ECL)detection system (Amersham Pharmacia, Piscataway, N.J.).

Example 11 Results with Abbreviated hGAA cDNA

High-level production of hGAA in muscle was shown with an AAV2/6 (AAV6)vector containing the shortened hGAA cDNA (FIG. 6). The hGAA level inskeletal muscle with the AAV6 vector is approximately 10-fold higherthan the GAA level in normal mice. The hGAA and glycogen content wasanalyzed in GAA-knockout (GAA-KO)/SCID mice that were treated at 6 weeksof age, so these levels reflect the delivery of hGAA in adult animals.The analysis was done 6 weeks following intramuscular AAV6 injection,and demonstrated a trend toward long-term expression. In addition,glycogen content was significantly reduced (p<0.002), indicating atherapeutically-relevant effect from this level of hGAA production inmuscle at that time point. Furthermore, prolonged hGAA expression andcomplete reduction of glycogen content to normal was observed in theinjected gastrocnemius muscle at 12-weeks after AAV6 vectoradministration (not shown).

These data indicate that intramuscular injection of AAV vector isefficacious in glycogen storage disease II.

We have further shown secretion from liver with the AAV2/6 (AAV6) andAAV2/2 (AAV2) versions of the vector containing the shortened hGAA cDNAin GAA-KO/SCID mice following portal vein injection (FIG. 7). Theabbreviated GAA vector was packaged within either an AAV2 or AAV6 capsidto generate two different vector stocks. Each stock was administered toa different mouse by portal vein injection. hGAA was detected in plasmafrom both mice by Western blot analysis. The data further demonstrateuptake of hGAA by skeletal muscle and heart with these vectors.

These results indicate that the liver can be used as a depot organ forhGAA production, with delivery to skeletal muscle and heart that resultsin a reduction in glycogen stores in these tissues due to uptake ofsecreted GAA.

Example 12 Intramuscular Versus Intraportal Delivery of AAV Vectors inGAA-KO Mice

An AAV2 vector packaged as AAV1 (AAV2/1) corrected glycogen storage wheninjected intramuscularly; however, the effect was observed only in theinjected muscle (Fraites et al. (2002) Mol. Ther. 5:571-578). On theother hand, when we administered an AAV vector by portal vein injectionin immunodeficient GAA-KO mice (GAA-KO/SCID mice) GAA was delivered toliver and other tissues (Sun et al. (2003) Mol. Ther. 2003; 7:467-477).Therefore, we investigated the difference in benefit betweenintramuscular and intraportal injection of the AAV vector, since thelatter approach delivered GAA to multiple target tissues includingskeletal muscles, the diaphragm and the heart.

We chose to evaluate the benefit of an AAV vector encoding the shortenedGAA targeted to skeletal muscle or to liver in the GAA-KO/SCID mousemodel. We administered an AAV vector encoding the shortened GAA byintramuscular or portal vein injection in GAA-KO/SCID mice, and analyzedGAA activity and glycogen content in tissues. The AAV2-derived vectorwas packaged as AAV2 (AAV2/2) or AAV6 (AAV2/6) for portal veininjections. Based on homology between the capsid proteins of AAV6 andAAV1, it was deemed likely that AAV2/6 would transduce myofibers moreefficiently than AAV2/2 (Chao et al. (2000) Mol. Ther. 2:619-623;Rabinowitz et al. (2002) J. Virol. 76:791-801; Rutledge et al. (1998) J.Virol. 72:309-319). The AAV2/6 vector was injected intramuscularly.Glycogen content and GAA activity were analyzed in tissues at 6, 12, and24 weeks after vector injection.

Results.

Following portal vein injection of an AAV2/2 vector, secreted GAA wasdetectable by Western blot analysis of plasma starting at 2 weeks andpersisted for 12 weeks (Data not shown). GAA activity was highlyelevated in multiple tissues compared to baseline levels in untreatedGAA-KO/SCID mice, and approximately 10-fold higher than wild-type levelsin the liver at 12 weeks following vector administration (FIG. 10).Human GAA was detected by Western blot analysis of multiple tissues,including the target tissues of heart and diaphragm (FIG. 11). Enzymeanalysis revealed that GAA activity was concordantly elevated in thesetissues (FIG. 10). These results demonstrated the secretion and uptakeof GAA following portal vein injection of the AAV vector, consistentwith the model of the liver as a depot organ for GAA production inGAA-deficient states as established with an Ad vector.

Following intramuscular injection with the AAV2/6 vector. GAA activityin the gastrocnemius muscle exceeded normal levels by approximately20-fold at 6, 12 and 24 weeks following vector administration (FIG. 12).In these mice the glycogen content in the injected muscle diminished tonear-normal levels for up to 24 weeks following vector administration,and decreased glycogen accumulation as evidenced by decreased stainingfor glycogen (FIG. 13). Despite the achievement of high-level GAAproduction in skeletal muscle, the GAA activity in other tissues was notmarkedly increased (FIG. 12).

Example 13 hGAA AAV Vectors with Altered Leader Signal Peptides

The peptide leader sequence (SEQ ID NO: 4, corresponding to amino acids1-27, SEQ ID NO: 2) of hGAA was replaced with the synthetic peptideleader sequence SP38 (Barash et al., (2002) Biochem. Biophys. Res. Comm.294:835-42), as well as peptide leader sequences from erythropoietin,albumin, alpha-1-antitrypsin and factor IX. The amino acid sequences ofthese leader peptides are shown in Table 2.

TABLE 2 Amino acid sequences for leader peptides Leader sequencePeptide sequence hGAA MGVRHPPCSHRLLAVCALVSLATAALL (SEQ ID NO: 4) SP38MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO: 5) Human erythropoietinMGVHECPAWLWLLLSLLSLPLGLPVLG (SEQ ID NO: 6) Human albuminMKWVTFISLLFLFSSAYS (SEQ ID NO: 7) Human alpha-1-MPSSVSWGILLLAGLCCLVPVLSA antitrypsin (SEQ ID NO: 8) Human coagulationMWRVNMIMAESPGLITICLLGYLLSAE factor IX CTVFLDHENANKILNRPKR (SEQ ID NO: 9)

Plasmid vectors that express hGAA in which the wild-type hGAA peptideleader sequence (SEQ ID NO: 4) is replaced with the different leaderpeptides listed in Table 2 were constructed using the plasmidAVCBhGAAG-delta as described briefly below. The resulting constructsencode a GAA peptide in winch one of the peptides of SEQ ID NOS: 5-9replaces the first 27 amino acids of SEQ ID NO: 1.

AVCBSP38GAAG-Delta Preparation Strategy.

A 0.37 Kb PCR product from AVCBhGAAG-delta was amplified using theprimers 5′-GCT GCA AAGCTT ggg cac atc cta ctc cat-3′ (SEQ ID NO: 10) and5′-ct gca gcc cct gct ttg cag gga tgt agc-3′ (SEQ ID NO: 11). Theresulting PCR product comprises nucleotide sequences that code for theregion of the hGAA protein immediately downstream from the native hGAAsignal peptide (nucleotides 523-796, SEQ ID NO: 1) and adds a HindIIIrestriction site at the signal peptide cleavage site. This PCR productwas digested with HindIII and SacII and gel-purified to produce DNAfragment I.

A second DNA fragment coding for the SP38 leader sequence,KpnI-SP38-HindIII, 5′-AGC TGC TGA GGTACC TCA GCC ACC atg tgg tgg cgc ctgtgg tgg ctg ctg ctg ctg ctg ctg ctg ctg tgg ccc atg gtg tgg gcc AAGCTTCGA TGC TAC GTC-3′, SEQ ID NO: 12, was hybridized with a reverse primer,5′-GAC GTA GCA TCG AAG CTT-3′, SEQ ID NO: 13. The resulting hybrid wasextended Klenow DNA polymerase in the presence of dNTPs to form adouble-stranded DNA fragment. The resulting DNA fragment contains a KpnIsite near the 5′ end followed by an optimal Kozak sequence (GCCACC) andthe SP38 leader peptide coding sequence, followed with a HindIII site atthe 3′ end of the SP38 sequence. This DNA was digested with KpnI andHindIII and gel-purified to produce DNA fragment II.

DNA fragments I and II described above were then ligated intoAVCBhGAAG-delta plasmid DNA digested with KpnI and SacII. The resultingligation mixture was used to transform STBL2 cells (Gibco-BRL), andtransformants containing AVCBSP38GAAG-delta were selected in thepresence of antibiotics and structure confirmed by gel electrophoresis.The resulting construct codes for a GAA peptide in which the hGAA leadersequence (SEQ ID NO: 4) has been replaced with the SP38 leader sequence(SEQ ID NO: 5).

A VCBSP-hEpoGAAG-delta preparation strategy. The plasmidAVCBSP-hEpoGAAG-delta was prepared in the same manner asAVCBSP38GAAG-delta, wherein fragment II was replaced with theKpnI-HindIII digestion product of the double-stranded DNA fragment withsequence 5′-AGC TGC TGA GGTACC TCA GCC ACC atgggggtg cacgaatgtcctgcctggct gtggcttctc ctgtccctgc tgtcgctccc tctgggcctc ccagtcctgg gcAAGCTT CGA TGC TAC GTC-3′ (SEQ ID NO: 14). The resulting construct codesfor a GAA peptide in which the hGAA leader sequence (SEQ ID NO: 4) hasbeen replaced with the hEPO leader sequence (SEQ ID NO: 6).

AVCBSPantitrypsinGAAG-Delta Preparation Strategy.

The plasmid AVCBSPantitrypsinGAAG-delta was prepared in the same manneras AVCBSP38GAAG-delta, using the KpnI-HindIII digestion product of thedouble-stranded DNA fragment with sequence 5′-C TGA GGTACC T GCC ACC-atgccgtcttct gtctcgtggg gcatcctcct gctggcaggc ctgtgctgcc tggtccctgtctccctggct-AAGCTT CGA T-3′ (SEQ ID NO: 15) in lieu of fragment II. Theresulting construct codes for a GAA peptide in which the hGAA leadersequence (SEQ ID NO: 4) has been replaced with the humanalpha-1-antityrpsin leader sequence (SEQ ID NO: 7).

AVCBSPALBGAAG-Delta Preparation Strategy.

AVCBSPALBGAAG-delta was prepared in the same manner asAVCBSP38GAAG-delta, using the KpnI-HindIII digestion product of thedouble-stranded DNA fragment with sequence 5′-C TGA GGTACC T GCC ACC-atgaagtgggt aacctttatt tcccttcttt ttctctttag ctcggcttat tcc-AAGCTT CGAT-3′ (SEQ ID NO: 16) in lieu of fragment II. The resulting constructcodes for a GAA peptide in which the hGAA leader sequence (SEQ ID NO: 4)has been replaced with the human albumin leader sequence (SEQ ID NO: 8).

AVCBSPFiXGAAG-Delta Preparation Strategy.

Lastly.

AVCBSPFIXGAAG-delta was prepared in the same manner asAVCBSP38GAAG-delta, using the KpnI-HindIII digestion product of thedouble-stranded DNA fragment with sequence 5′-C TGA GGTACC T GCC ACCatgcagcgcg tgaacatgat catggca-3′ (SEQ ID NO: 17) in lieu of fragment II.The resulting construct codes for a GAA peptide in which the hGAA leadersequence (SEQ ID NO: 4) has been replaced with the human factor IXleader sequence (SEQ ID NO: 9).

The resulting plasmids were used to examine the effect of differentleader sequences on the localization of hGAA in cells transfected withthese plasmids.

Example 14 Relative Secretion of hGAA with Altered Leader Sequences in293 Cells

293 cells were transfected with AAV vector plasmids described in Example13 and were collected 40 hours post-transfection. Total hGAA activitywas assayed both in the cells and in the medium. These results aredepicted in FIG. 14 and the relative hGAA secretion is summarized inTable 3.

TABLE 3 hGAA secretion with different leader sequences Proportion hGAA %Total Increased Leader secreted (hGAA hGAA hGAA sequence medium/hGAAcells) secreted secretion (fold) HGAA 0.53 34 N/A SP38.1 9.7 91 18 Epo1.1 53 2.1 α-1-antitrypsin 8.5 90 16 Factor IX 14 93 26

Western blot analysis of cellular and secreted hGAA in 293 cellstransfected with the AAV vector plasmids described in Example 13demonstrated normal migration, consistent with unaltered glycosylationand processing of chimeric hGAA linked to alternative signal peptides(FIG. 15). This data supported the normal glycosylation of hGAA, despitethe increased secretion and presumably shortened residence in the Golgi(Wisselaar et al., (1993) J. Biol. Chem 268 (3): 2223-31). For furtherevidence of the normal glycosylation of hGAA produced with theseconstructs, we injected mice with the AAV2/8 vector encoding thechimeric α-1-antitrypsin signal peptide linked to the hGAA cDNA (minusthe 27 amino acid GAA signal peptide), and hGAA was detectedcorresponding to the ˜110 kD hGAA precursor (FIG. 16). The hGAA levelwas lower for female mice (lanes 5-7 and 11-13), as expected given lowertransduction with AAV2/8 vectors in female mice. Significantly, hGAAsecretion was higher for the vector containing the chimericα-1-antitrypsin signal peptide (lanes 2-7), than for the vectorcontaining the hGAA signal peptide (lanes 8-13).

Tissue GAA activity was increased in tissues for the 3 male GAA-KO/SCIDmice that received the AAV vector encoding the alpha-1-antitrypsinsignal peptide linked to hGAA (corresponding to lanes 2-5 in FIG. 16).Significantly, liver GAA activity remained at near normal levels,despite increased GAA activity in tissues compared to controls (FIG.17). These results were obtained at only 2 weeks following vectoradministration, as opposed to the later 6-week time point used in theother experiments, and indicated early uptake of secreted GAA in thetarget tissues of heart, diaphragm, and at lower levels, skeletalmuscle. GAA uptake in target tissues is expected to increase after the2-week time point, based on previous results. Finally, a Western blot ofthe target tissues showed appropriate processing of hGAA to ˜76 kD and˜67 kD (data not shown). These data support the hypothesis that chimerichGAA linked to alternative signal peptides will be appropriatelysecreted, processed and targeted to lysosomes in target tissues, despitethe maintenance of normal GAA levels in liver. This novel developmentwill reduce any potential toxicity for overexpression of hGAA in thedepot organ, the liver, in contrast to all previous approaches toliver-targeted gene therapy in Pompe disease that produced extremesupra-physiological levels of GAA in the liver

Example 15 Expression of hGAA with a Liver-Specific Promoter inImmunocompetent GAA-KO Mice

An AAV2/8 vector encoding hGAA driven by a liver-specific promoter (WangL et al., (1999) Proc Nat Acad Sci USA 96:3906-10) was administeredintravenously in GAA-KO mice. Contrary to previous experiments, wherehGAA was eliminated in plasma by anti-GAA antibodies in immunocompetentGAA-KO mice, hGAA persisted in plasma as detected by Western blotanalysis (FIG. 18). Therefore, the limitation of GAA expression to theliver eliminated the antibody response and allowed persistent GAAsecretion with implications for gene therapy in Pompe disease.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A vector comprising a nucleic acid encoding a chimeric polypeptide comprising a secretory signal sequence operably linked to a lysosomal polypeptide, wherein said secretory signal sequence replaces the native secretory signal sequence of the lysosomal polypeptide.
 2. The vector of claim 1, wherein said nucleic acid is operatively linked to a transcriptional control element operable in liver cells.
 3. The vector of claim 1, wherein the secretory signal sequence is derived from a secreted polypeptide.
 4. The vector of claim 1, wherein the secretory signal sequence is derived from SP38, erythropoietin albumin, albumin, or coagulation factor IX.
 5. The vector of claim 1, wherein the secretory signal sequence comprises the amino acid sequence set forth in any of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 9, or an amino acid sequence having at least 98% sequence identity to the sequence set forth in any of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO:
 9. 6. The vector of claim 1, wherein the vector is an adeno-associated virus (AAV).
 7. The vector of claim 1, wherein secretory signal sequence replaces the amino terminus of the native lysosomal polypeptide
 8. A pharmaceutical formulation comprising the vector of claim 1 in a pharmaceutically acceptable carrier.
 9. An isolated cell comprising the vector of claim
 1. 10. An in-vitro method of delivering a nucleic acid encoding a lysosomal polypeptide to a cell, comprising contacting a cell in vitro with the vector according to claim 1 under conditions sufficient for the vector to be introduced into the cell and for the nucleic acid to be expressed to produce the chimeric polypeptide comprising the secretory signal sequence operably linked to the lysosomal polypeptide wherein said secretory signal sequence replaces the native secretory signal sequence of the lysosomal polypeptide.
 11. The method of claim 10, wherein the cell is a cultured cell.
 12. The vector of claim 10, wherein the secretory signal sequence is derived from SP38, erythropoietin albumin, albumin, or coagulation factor IX.
 13. A method of producing a lysosomal polypeptide in a cultured cell, comprising: contacting a cultured cell with the vector according to claim 1 under conditions sufficient for the vector to be introduced into the cultured cell and for the nucleic acid to be expressed to produce the chimeric polypeptide comprising the secretory signal operably linked to the polypeptide wherein said secretory signal sequence replaces the native secretory signal sequence of the lysosomal polypeptide and the polypeptide is secreted from the cultured, and collecting the polypeptide secreted onto the cell culture medium.
 14. The method of claim 13, wherein the cell is a mammalian cell.
 15. The method of claim 13, wherein the cell is a CHO cell, a 293 cell, a HT1080 cell, a HeLa cell or a C10 cell.
 16. The method of claim 13, wherein the secretory signal sequence is derived from SP38, erythropoietin albumin, albumin, or coagulation factor IX.
 17. An in vitro method of delivering a nucleic acid encoding a lysosomal polypeptide to a cell, comprising contacting a cell in vitro with the vector according to claim 1 under conditions sufficient for the vector to be introduced into the cell and for the nucleic acid to be expressed to produce the chimeric polypeptide comprising the secretory signal sequence operably linked to the lysosomal polypeptide wherein said secretory signal sequence replaces the native secretory signal sequence of the lysosomal polypeptide.
 18. The method of claim 17, wherein the secretory signal sequence is derived from SP38, erythropoietin albumin, albumin, or coagulation factor IX.
 19. A method of treating a deficiency of a lysosomal polypeptide in a subject, comprising administering to the subject the pharmaceutical formulation of claim 8 in a therapeutically effective amount.
 20. The method of claim 19, wherein the secretory signal sequence is derived from SP38, erythropoietin albumin, albumin, or coagulation factor IX. 